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Sample records for proton-translocating atpases enhanced

  1. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

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    Summer R. Hayek

    2014-01-01

    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

  2. Enhanced Proton Translocating Pyrophosphatase Activity Improves Nitrogen Use Efficiency in Romaine Lettuce1[C][W][OA

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    Paez-Valencia, Julio; Sanchez-Lares, Jonathan; Marsh, Ellen; Dorneles, Liane T.; Santos, Mirella P.; Sanchez, Diego; Winter, Alexander; Murphy, Sean; Cox, Jennifer; Trzaska, Marcin; Metler, Jason; Kozic, Alex; Facanha, Arnoldo R.; Schachtman, Daniel; Sanchez, Charles A.; Gaxiola, Roberto A.

    2013-01-01

    Plant nitrate (NO3−) acquisition depends on the combined activities of root high- and low-affinity NO3− transporters and the proton gradient generated by the plasma membrane H+-ATPase. These processes are coordinated with photosynthesis and the carbon status of the plant. Here, we present the characterization of romaine lettuce (Lactuca sativa ‘Conquistador’) plants engineered to overexpress an intragenic gain-of-function allele of the type I proton translocating pyrophosphatase (H+-PPase) of Arabidopsis (Arabidopsis thaliana). The proton-pumping and inorganic pyrophosphate hydrolytic activities of these plants are augmented compared with control plants. Immunohistochemical data show a conspicuous increase in H+-PPase protein abundance at the vasculature of the transgenic plants. Transgenic plants displayed an enhanced rhizosphere acidification capacity consistent with the augmented plasma membrane H+-ATPase proton transport values, and ATP hydrolytic capacities evaluated in vitro. These transgenic lines outperform control plants when challenged with NO3− limitations in laboratory, greenhouse, and field scenarios. Furthermore, we report the characterization of a lettuce LsNRT2.1 gene that is constitutive up-regulated in the transgenic plants. Of note, the expression of the LsNRT2.1 gene in control plants is regulated by NO3− and sugars. Enhanced accumulation of 15N-labeled fertilizer by transgenic lettuce compared with control plants was observed in greenhouse experiments. A negative correlation between the level of root soluble sugars and biomass is consistent with the strong root growth that characterizes these transgenic plants. PMID:23307651

  3. Sodium ions as substitutes for protons in the gastric H,K-ATPase

    International Nuclear Information System (INIS)

    Polvani, C.; Sachs, G.; Blostein, R.

    1989-01-01

    In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes

  4. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

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    Schep, Daniel G.; Rubinstein, John L.

    2016-01-01

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  5. On Allosteric Modulation of P-Type Cu+-ATPases

    DEFF Research Database (Denmark)

    Mattle, Daniel; Sitsel, Oleg; Autzen, Henriette Elisabeth

    2013-01-01

    P-type ATPases perform active transport of various compounds across biological membranes and are crucial for ion homeostasis and the asymmetric composition of lipid bilayers. Although their functional cycle share principles of phosphoenzyme intermediates, P-type ATPases also show subclass...... of intramembranous Cu+ binding, and we suggest an alternative role for the proposed second site in copper translocation and proton exchange. The class-specific features demonstrate that topological diversity in P-type ATPases may tune a general energy coupling scheme to the translocation of compounds with remarkably...

  6. MODULATION OF GROWTH AND PROTON PUMPING ATPase ACTIVITY OF PROBIOTIC Lactobacilli BY DIETARY CUCURBITS

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    Irfan Ahmad

    2013-12-01

    Full Text Available Gastrointestinal tract predominantly harbor probiotic Lactobacilli which exert beneficial effects on human health. Aqueous extracts from fruits of Lagenaria siceraria (Ls, Luffa cylindrica (Lc and Cucurbita maxima (Cm were prepared and lyophilized. Fruit extracts were investigated for their effects on Lactobacillus rhamnosus (L. rhamnosus, Lactobacillus plantarum (L. plantarum and Lactobacillus acidophilus (L. acidophilus. Extracts were found to enhance growth of Lactobacilli without any toxic effect (up to 1000µg/mL concentration. Minimum concentration of extracts at which growth of probiotic strains were found to be enhanced significantly were determined (103.67 µg/mL-118µg/mL and considered as effective concentration (EC or growth stimulatory concentration (GSC. Proton pumping ATPase activity of Lactobacilli were examined and found to be enhanced significantly (29.89- 61.96% in extracts treated probiotics (Lactobacilli as compared to the normal control. Inulin used as positive control and found to enhance the proton efflux activity (28.06-37.72% with respect to the control. These dietary cucurbits enhance metabolic activity of probiotic Lactobacilli by modulating their proton pumping ATPase mechanism. This study suggested that the consumption of cucurbit fruits might be a natural source of enhancing the activities of probiotic Lactobacilli in the gut.

  7. Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver

    International Nuclear Information System (INIS)

    Yeh, H.I.; van Rossum, G.D.

    1991-01-01

    We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles

  8. Plant P4-ATPases: lipid translocators with a role in membrane traficking

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    a large family of membrane proteins involved in pumping different physiologically-relevant substrates across biological membranes [4]. The members of the P4 subfamily (also known as flippases) catalyze the energy-driven translocation of lipids necessary for establishing transbilayer lipid asymmetry [5......], a feature necessary for correct functioning of the cells [6,7]. Deletion of one or more P4-ATPase genes causes defects in vesicle budding in various organisms [8-10] and some members of the yeast family have been shown to interact with the vesiculation machinery [11,12]. Thus, unraveling the key features...... of P4-ATPase functioning is crucial to understand the mechanisms underlying the whole secretory and endocytic pathways. In the model plant Arabidopsis, 12 members of the P4-ATPase family have been described (ALA1-ALA12, for Aminophospholipid ATPase) [4]. In the past years, we have characterized several...

  9. Porphyromonas gingivalis is highly sensitive to inhibitors of a proton-pumping ATPase.

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    Sekiya, Mizuki; Shimoyama, Yu; Ishikawa, Taichi; Sasaki, Minoru; Futai, Masamitsu; Nakanishi-Matsui, Mayumi

    2018-04-15

    Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

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    Tadini-Buoninsegni, Francesco; Moncelli, Maria Rosa; Peruzzi, Niccolò; Ninham, Barry W.; Dei, Luigi; Nostro, Pierandrea Lo

    2015-10-01

    The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na+, K+-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.

  11. The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump.

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    Moriyama, Y; Nelson, N

    1987-07-05

    The proton-ATPase of chromaffin granules was purified so as to maintain its proton-pumping activity when reconstituted into phospholipid vesicles. The purification procedure involved solubilization with polyoxyethylene 9 lauryl ether, hydroxylapatite column, precipitation by ammonium sulfate, and glycerol gradient centrifugation. The protease inhibitor mixture used in previous studies inhibited the proton-pumping activity of the enzyme; therefore, the protein was stabilized by pepstatin A and leupeptin. The enzyme was purified at least 50-fold with respect to both ATPase and proton-pumping activity. The ATP-dependent proton uptake activity of the reconstituted enzyme was absolutely dependent on the presence of Cl- or Br- outside the vesicles, whereas sulfate, acetate, formate, nitrate, and thiocyanate were inhibitory. Sulfate inhibition seems to be due to competition with Cl- on the anion-binding site outside the vesicles, whereas nitrate and thiocyanate inhibited only from the internal side. As with the inhibition by N-ethylmaleimide, the proton-pumping activity was much more sensitive to nitrate than the ATPase activity. About 20 mM nitrate were sufficient for 90% inhibition of the proton-pumping activity while 100 mM inhibited only 50% of the ATPase activity both in situ and in the reconstituted enzyme. The possible regulatory effect of anions on the ATP-dependent proton uptake in secretory granules is discussed.

  12. Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase

    International Nuclear Information System (INIS)

    Ceccarelli, E.; Vallejos, R.H.

    1983-01-01

    Two different types of essential carboxyl groups were detected in the extrinsic component of the proton ATPase of Rhodospirillum rubrum. Chemical modification of R. rubrum chromatophores or its solubilized ATPase by Woodward's reagent K resulted in inactivation of photophosphorylating and ATPase activities. The apparent order of reaction was nearly 1 with respect to reagent concentration and similar K1 were obtained for the soluble and membrane-bound ATPases suggesting that inactivation was associated with modification of one essential carboxyl group located in the soluble component of the proton ATPase. Inactivation was prevented by adenine nucleotides but not by divalent cations. Dicyclohexylcarbodiimide completely inhibited the solubilized ATPase with a K1 of 5.2 mM and a K2 of 0.81 min-1. Mg2+ afforded nearly complete protection with a Kd of 2.8 mM. Two moles of [14C]dicyclohexylcarbodiimide were incorporated per mole of enzyme for complete inactivation but in the presence of 30 mM MgCl2 only one mole was incorporated and there was no inhibition. The labeling was recovered mostly from the beta subunit. The incorporation of the labeled reagent into the ATPase was not prevented by previous modification with Woodward's reagent K. It is concluded that both reagents modified two different essential carboxyl groups in the soluble ATPase from R. rubrum

  13. Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Harper, J.F.; Surowy, T.K.; Sussman, M.R.

    1989-01-01

    In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H + -ATPase) that produces an electric potential and pH gradient. The authors isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H + -ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plant contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops

  14. Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

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    Stransky, Laura A.; Forgac, Michael

    2015-01-01

    The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

  15. An Arginine Finger Regulates the Sequential Action of Asymmetrical Hexameric ATPase in the Double-Stranded DNA Translocation Motor.

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    Zhao, Zhengyi; De-Donatis, Gian Marco; Schwartz, Chad; Fang, Huaming; Li, Jingyuan; Guo, Peixuan

    2016-10-01

    Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation and virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures. The isolated dimer alone was inactive in DNA translocation, but the addition of monomer could restore the activity, suggesting that the hexameric ATPase ring contained both dimer and monomers. Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of the ATPase with high or low DNA affinity. Taking these observations together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The finding of asymmetrical hexameric organization is supported by structural evidence of many other ATPase systems showing the presence of one noncovalent dimer and four monomer subunits. All of these provide clues for why the asymmetrical hexameric ATPase gp16 of ϕ29 was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM) since the contact by the arginine finger renders two adjacent ATPase subunits closer than other subunits. Thus, the asymmetrical hexamer would appear as a pentamer by cryo-EM, a technology that acquires the average of many images. Copyright © 2016 Zhao et al.

  16. Hemin reconstitutes proton extrusion in an H+-ATPase-negative mutant of Lactococcus lactis

    DEFF Research Database (Denmark)

    Blank, L.M.; Købmann, Brian Jensen; Michelsen, Ole

    2001-01-01

    H+-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H+-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells...

  17. Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis.

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    Roy, Jeremy W; Hill, Eric; Ruan, Ye Chun; Vedovelli, Luca; Păunescu, Teodor G; Brown, Dennis; Breton, Sylvie

    2013-08-15

    Clear cells express the vacuolar proton-pumping H(+)-ATPase (V-ATPase) and acidify the lumen of the epididymis, a process that is essential for male fertility. The renin-angiotensin-aldosterone system (RAAS) regulates fluid and electrolyte balance in the epididymis, and a previous study showed binding of aldosterone exclusively to epididymal clear cells (Hinton BT, Keefer DA. Steroid Biochem 23: 231-233, 1985). We examined here the role of aldosterone in the regulation of V-ATPase in the epididymis. RT-PCR showed expression of the mineralocorticoid receptor [MR; nuclear receptor subfamily 3, group C member 2 (NR3C2)] and 11-β-dehydrogenase isozyme 2 (HSD11β2) mRNAs specifically in clear cells, isolated by fluorescence-activated cell sorting from B1-enhanced green fluorescent protein (EGFP) mice. Tail vein injection of adult rats with aldosterone, 1,2-dioctanoyl-sn-glycerol (DOG), or 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) induced V-ATPase apical membrane accumulation and extension of V-ATPase-labeled microvilli in clear cells in the caput epididymis but not in the cauda. V-ATPase activity was measured in EGFP-expressing clear cells using the intracellular pH (pHi)-sensing dye seminaphthorhodafluor-5F-5-(and 6)-carboxylic acid, acetoxymethyl ester acetate (SNARF-5F). Aldosterone induced a rapid increase in the rate of Na(+)- and bicarbonate-independent pHi recovery following an NH4Cl-induced acid load in clear cells isolated from the caput but not the cauda. This effect was abolished by concanamycin A, spironolactone, and chelerythrine but not myristoylated-protein kinase inhibitor (mPKI) or mifepristone. Thus aldosterone increases V-ATPase-dependent proton secretion in clear cells in the caput epididymis via MR/NR3C2 and PKC activation. This study, therefore, identifies aldosterone as an active member of the RAAS for the regulation of luminal acidification in the proximal epididymis.

  18. Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

    Science.gov (United States)

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg

    2014-01-01

    ) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4......Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases...... to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell....

  20. The Presynaptic v-ATPase Reversibly Disassembles and Thereby Modulates Exocytosis but Is Not Part of the Fusion Machinery

    Directory of Open Access Journals (Sweden)

    Anna Bodzęta

    2017-08-01

    Full Text Available Vacuolar H+-ATPase (v-ATPase is a multi-subunit complex comprising two domains: the cytosolic V1 domain catalyzing ATP hydrolysis and the membranous V0 sector translocating protons across membranes. In addition to proton pumping, a direct function of the V0 proteolipid ring in membrane fusion has been proposed for yeast vacuolar fusion and synaptic vesicle exocytosis in Drosophila. Here, we show in cultured hippocampal neurons that in recycling synaptic vesicles, v-ATPases are only transiently assembled in a pH-dependent fashion during the tightly coupled cycle of exo-endocytosis. Upon locking v-ATPase in an assembled state by saliphenylhalamide, we observed use- and time-dependent release depression for stimuli exceeding release of primed vesicles but no abrogation of exocytosis. Thus, the membranous V0 sector is not part of the exocytotic fusion machinery. Instead, v-ATPase modulates release upstream of docking to favor fusion of fully filled synaptic vesicles.

  1. The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states.

    Science.gov (United States)

    Ray, Tushar

    2013-01-01

    This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

  2. Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

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    Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

    2010-02-01

    We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

  3. Anticancer ruthenium(III) complex KP1019 interferes with ATP-dependent Ca2+ translocation by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA).

    Science.gov (United States)

    Sadafi, Fabrizio-Zagros; Massai, Lara; Bartolommei, Gianluca; Moncelli, Maria Rosa; Messori, Luigi; Tadini-Buoninsegni, Francesco

    2014-08-01

    Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a P-type ATPase that sustains Ca2+ transport and plays a major role in intracellular Ca2+ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium-based anticancer drugs, namely KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]), NAMI-A (imidazolium [trans-tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)]) and RAPTA-C ([Ru(η6-p-cymene)dichloro(1,3,5-triaza-7-phosphaadamantane)]), and cisplatin (cis-diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid-supported membrane was measured after ATP or Ca2+ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP-dependent translocation of Ca2+ ions. An IC50 value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca2+ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

  5. AAA-ATPases in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Ravikiran S. Yedidi

    2017-06-01

    Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  6. AAA-ATPases in Protein Degradation.

    Science.gov (United States)

    Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

    2017-01-01

    Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  7. Chemiosmotic Energy Conservation in Dinoroseobacter shibae: Proton Translocation Driven by Aerobic Respiration, Denitrification, and Photosynthetic Light Reaction

    Directory of Open Access Journals (Sweden)

    Christian Kirchhoff

    2018-05-01

    Full Text Available Dinoroseobacter shibae is an aerobic anoxygenic phototroph and able to utilize light energy to support its aerobic energy metabolism. Since the cells can also grow anaerobically with nitrate and nitrite as terminal electron acceptor, we were interested in how the cells profit from photosynthesis during denitrification and what the steps of chemiosmotic energy conservation are. Therefore, we conducted proton translocation experiments and compared O2-, NO3-, and NO2- respiration during different light regimes and in the dark. We used wild type cells and transposon mutants with knocked-out nitrate- and nitrite- reductase genes (napA and nirS, as well as a mutant (ppsR impaired in bacteriochlorophyll a synthesis. Light had a positive impact on proton translocation, independent of the type of terminal electron acceptor present. In the absence of an electron acceptor, however, light did not stimulate proton translocation. The light-driven add-on to proton translocation was about 1.4 H+/e- for O2 respiration and about 1.1 H+/e- for NO3- and NO2-. We could see that the chemiosmotic energy conservation during aerobic respiration involved proton translocation, mediated by the NADH dehydrogenase, the cytochrome bc1 complex, and the cytochrome c oxidase. During denitrification the last proton translocation step of the electron transport was missing, resulting in a lower H+/e- ratio during anoxia. Furthermore, we studied the type of light-harvesting and found that the cells were able to channel light from the green–blue spectrum most efficiently, while red light has only minor impact. This fits well with the depth profiles for D. shibae abundance in the ocean and the penetration depth of light with different wavelengths into the water column.

  8. Characterization of a heavy metal translocating P-type ATPase gene from an environmental heavy metal resistance Enterobacter sp. isolate.

    Science.gov (United States)

    Chien, Chih-Ching; Huang, Chia-Hsuan; Lin, Yi-Wei

    2013-03-01

    Heavy metals are common contaminants found in polluted areas. We have identified a heavy metal translocating P-type ATPase gene (hmtp) via fosmid library and in vitro transposon mutagenesis from an Enterobacter sp. isolate. This gene is believed to participate in the bacterium's heavy metal resistance traits. The complete gene was identified, cloned, and expressed in a suitable Escherichia coli host cell. E. coli W3110, RW3110 (zntA::Km), GG48 (ΔzitB::Cm zntA::Km), and GG51 (ΔzitB::Cm) were used to study the possible effects of this gene for heavy metal (cadmium and zinc in particular) resistance. Among the E. coli strains tested, RW3110 and GG48 showed more sensitivity to cadmium and zinc compared to the wild-type E. coli W3110 and strain GG51. Therefore, strains RW3110 and GG48 were chosen for the reference hosts for further evaluation of the gene's effect. The results showed that expression of this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms.

  9. Proton Pumping and Slippage Dynamics of a Eukaryotic P-Type ATPase Studied at the Single-Molecule Level

    DEFF Research Database (Denmark)

    Veshaguri, Salome

    In all eukaryotes the plasma membrane potential and secondary transport systems are energized by P-type ATPases whose regulation however remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform....... We propose that variable ATP/H+ stoichiometry emerges as a novel mechanism for adaptation when challenged with depletion of ATP that is likely relevant for other ATPases. Such measurements will provide indispensable insights into the mechanisms of function and regulation of many other ion...

  10. Cloning, expression, purification, crystallization and initial crystallographic analysis of the preprotein translocation ATPase SecA from Thermus thermophilus

    International Nuclear Information System (INIS)

    Vassylyeva, Marina N.; Mori, Hiroyuki; Tsukazaki, Tomoya; Yokoyama, Shigeyuki; Tahirov, Tahir H.; Ito, Koreaki; Vassylyev, Dmitry G.

    2006-01-01

    The SecA ATPase from T. thermophilus was cloned, expressed, purified and crystallized. Complete diffraction data sets were collected for two crystal forms at 2.8 and 3.5 Å resolution, respectively. Determination of the structure is now in progress. The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3 1(2) 21 (a = b = 168.6, c = 149.8 Å) and P6 1(5) 22 (a = b = 130.9, c = 564.6 Å). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 Å resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress

  11. V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

    NARCIS (Netherlands)

    Jansen, E.J.S.; Hafmans, T.G.M.; Martens, G.J.

    2010-01-01

    The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit

  12. The gastric H, K-ATPase system also functions as the Na, K-ATPase and Ca-ATPase in altered states [v1; ref status: indexed, http://f1000r.es/1eo

    Directory of Open Access Journals (Sweden)

    Tushar Ray

    2013-07-01

    Full Text Available This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump and/or Ca-ATPase (Ca-pump depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM fraction exhibits a (Ca or Mg-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF, the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

  13. P4 ATPases: Flippases in Health and Disease

    Directory of Open Access Journals (Sweden)

    Coen C. Paulusma

    2013-04-01

    Full Text Available P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed “lipid flipping”. Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.

  14. Protons and how they are transported by proton pumps

    DEFF Research Database (Denmark)

    Buch-Pedersen, Morten Jeppe; Pedersen, Bjørn Panyella; Veierskov, Bjarke

    2008-01-01

    The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. The recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Taking the biochemical and structural data together, we are now able to describe the basic...... molecular components that allow the plasma membrane proton H(+)-ATPase to carry out proton transport against large membrane potentials. When divergent proton pumps such as the plasma membrane H(+)-ATPase, bacteriorhodopsin, and F(O)F(1) ATP synthase are compared, unifying mechanistic premises for biological...

  15. The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states [v2; ref status: indexed, http://f1000r.es/1tc

    Directory of Open Access Journals (Sweden)

    Tushar Ray

    2013-09-01

    Full Text Available This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump and/or Ca-ATPase (Ca-pump depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM fraction exhibits a (Ca or Mg-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF, the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

  16. The Arabidopsis P4-ATPase ALA3 requires a ß-subunit to function in phospholipid translocation and secretory vesicle formation

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    The Arabidopsis P4-ATPase ALA3 requires a ß-subunit to function in phospholipid translocation and secretory vesicle formation   Lisbeth R. Poulsen1, Rosa L. López-Marqués1, Stephen C. McDowell2, Juha Okkeri3, Dirk Licht3, Alexander Schulz1, Thomas Pomorski3,  Jeffrey F. Harper2, and Michael G....... Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Department of Plant Biology, University of Copenhagen, DK-1871 Frederiksberg C, Denmark 2Biochemistry Department MS200, University of Nevada Reno, NV 89557, USA 3Humboldt-University Berlin, Faculty...... and in inducing membrane curvature, which is a requirement for vesicle formation. We show that Aminophospholipid ATPase3 (ALA3), a member of the P4-ATPase subfamily in the plant Arabidopsis thaliana, localizes to the Golgi apparatus and that genetic lesions of ALA3 result in impaired growth of roots and shoots...

  17. V-ATPase is a candidate therapeutic target for Ewing sarcoma.

    Science.gov (United States)

    Avnet, Sofia; Di Pompo, Gemma; Lemma, Silvia; Salerno, Manuela; Perut, Francesca; Bonuccelli, Gloria; Granchi, Donatella; Zini, Nicoletta; Baldini, Nicola

    2013-08-01

    Suppression of oxidative phosphorylation combined with enhanced aerobic glycolysis and the resulting increased generation of protons are common features of several types of cancer. An efficient mechanism to escape cell death resulting from intracellular acidification is proton pump activation. In Ewing sarcoma (ES), although the tumor-associated chimeric gene EWS-FLI1 is known to induce the accumulation of hypoxia-induced transcription factor HIF-1α, derangements in metabolic pathways have been neglected so far as candidate pathogenetic mechanisms. In this paper, we observed that ES cells simultaneously activate mitochondrial respiration and high levels of glycolysis. Moreover, although the most effective detoxification mechanism of proton intracellular storage is lysosomal compartmentalization, ES cells show a poorly represented lysosomal compartment, but a high sensitivity to the anti-lysosomal agent bafilomycin A1, targeting the V-ATPase proton pump. We therefore investigated the role of V-ATPase in the acidification activity of ES cells. ES cells with the highest GAPDH and V-ATPase expression also showed the highest acidification rate. Moreover, the localization of V-ATPase was both on the vacuolar and the plasma membrane of all ES cell lines. The acidic extracellular pH that we reproduced in vitro promoted high invasion ability and clonogenic efficiency. Finally, targeting V-ATPase with siRNA and omeprazole treatments, we obtained a significant selective reduction of tumor cell number. In summary, glycolytic activity and activation of V-ATPase are crucial mechanisms of survival of ES cells and can be considered as promising selective targets for the treatment of this tumor. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Drug Repositioning of Proton Pump Inhibitors for Enhanced Efficacy and Safety of Cancer Chemotherapy

    Directory of Open Access Journals (Sweden)

    Kenji Ikemura

    2017-12-01

    Full Text Available Proton pump inhibitors (PPIs, H+/K+-ATPase inhibitors, are the most commonly prescribed drugs for the treatment of gastroesophageal reflux and peptic ulcer diseases; they are highly safe and tolerable. Since PPIs are frequently used in cancer patients, studies investigating interactions between PPIs and anticancer agents are of particular importance to achieving effective and safe cancer chemotherapy. Several studies have revealed that PPIs inhibit not only the H+/K+-ATPase in gastric parietal cells, but also the vacuolar H+-ATPase (V-ATPase overexpressed in tumor cells, as well as the renal basolateral organic cation transporter 2 (OCT2 associated with pharmacokinetics and/or renal accumulation of various drugs, including anticancer agents. In this mini-review, we summarize the current knowledge regarding the impact of PPIs on the efficacy and safety of cancer chemotherapeutics via inhibition of targets other than the H+/K+-ATPase. Co-administration of clinical doses of PPIs protected kidney function in patients receiving cisplatin and fluorouracil, presumably by decreasing accumulation of cisplatin in the kidney via OCT2 inhibition. In addition, co-administration or pretreatment with PPIs could inhibit H+ transport via the V-ATPase in tumor cells, resulting in lower extracellular acidification and intracellular acidic vesicles to enhance the sensitivity of the tumor cells to the anticancer agents. In the present mini-review, we suggest that PPIs enhance the efficacy and safety of anticancer agents via off-target inhibition (e.g., of OCT2 and V-ATPase, rather than on-target inhibition of the H+/K+-ATPase. The present findings should provide important information to establish novel supportive therapy with PPIs during cancer chemotherapy.

  19. P4 ATPases - lipid flippases and their role in disease

    NARCIS (Netherlands)

    Folmer, Dineke E.; Elferink, Ronald P. J. Oude; Paulusma, Coen C.

    2009-01-01

    P4 ATPases (type 4 P-type ATPases) are multispan transmembrane proteins that have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Studies in Saccharomyces cerevisiae have indicated that P4 ATPases are important in vesicle

  20. Evolutionary primacy of sodium bioenergetics

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2008-04-01

    Full Text Available Abstract Background The F- and V-type ATPases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of ATP. Both the F-type (found in most bacteria and eukaryotic mitochondria and chloroplasts and V-type (found in archaea, some bacteria, and eukaryotic vacuoles ATPases can translocate either protons or sodium ions. The prevalent proton-dependent ATPases are generally viewed as the primary form of the enzyme whereas the sodium-translocating ATPases of some prokaryotes are usually construed as an exotic adaptation to survival in extreme environments. Results We combine structural and phylogenetic analyses to clarify the evolutionary relation between the proton- and sodium-translocating ATPases. A comparison of the structures of the membrane-embedded oligomeric proteolipid rings of sodium-dependent F- and V-ATPases reveals nearly identical sets of amino acids involved in sodium binding. We show that the sodium-dependent ATPases are scattered among proton-dependent ATPases in both the F- and the V-branches of the phylogenetic tree. Conclusion Barring convergent emergence of the same set of ligands in several lineages, these findings indicate that the use of sodium gradient for ATP synthesis is the ancestral modality of membrane bioenergetics. Thus, a primitive, sodium-impermeable but proton-permeable cell membrane that harboured a set of sodium-transporting enzymes appears to have been the evolutionary predecessor of the more structurally demanding proton-tight membranes. The use of proton as the coupling ion appears to be a later innovation that emerged on several independent occasions. Reviewers This article was reviewed by J. Peter Gogarten, Martijn A. Huynen, and Igor B. Zhulin. For the full reviews, please go to the Reviewers' comments section.

  1. Is the ATPase from halobacterium saccharovorum an evolutionary relic?

    Science.gov (United States)

    Hochstein, L. I.; Altekar, W.; Kristjansson, H.

    1986-01-01

    The ATP Synthase Complex present in the membranes of mitochondria, chloroplasts or bacteria is composed of 2 sectors: FO, an integral membrane protein consisting of 3 subunits mediating proton translocation across the membrane and F1, the catalytic component composed of 5 non-identical subunits. The apparent early origin of the ATP Synthase Complex, as implied by its ubiquitous distribution, seems inconsistent with its structural and functional complexity and raises the question if simpler versions of the ATP Synthase exist. Such an ATP Synthase has been searched for in various Archaebacteria. A purified halobacterial ATPase activity which possesses certain properties consistent with those of an ATP Synthase but which has a different subunit structure is described.

  2. Protons and how they are transported by proton pumps

    DEFF Research Database (Denmark)

    Buch-Pedersen, Morten Jeppe; Pedersen, Bjørn Panyella; Nissen, Poul

    2008-01-01

    molecular components that allow the plasma membrane proton H(+)-ATPase to carry out proton transport against large membrane potentials. When divergent proton pumps such as the plasma membrane H(+)-ATPase, bacteriorhodopsin, and F(O)F(1) ATP synthase are compared, unifying mechanistic premises for biological...... proton pumps emerge. Most notably, the minimal pumping apparatus of all pumps consists of a central proton acceptor/donor, a positively charged residue to control pK (a) changes of the proton acceptor/donor, and bound water molecules to facilitate rapid proton transport along proton wires....

  3. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  4. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase...... maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...

  5. The mechanism of coupling between oxido-reduction and proton translocation in respiratory chain enzymes.

    Science.gov (United States)

    Papa, Sergio; Capitanio, Giuseppe; Papa, Francesco

    2018-02-01

    The respiratory chain of mitochondria and bacteria is made up of a set of membrane-associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane-associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co-operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X-ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone-cytochrome c oxidoreductase and NADH-ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes. © 2017 Cambridge Philosophical Society.

  6. Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexycarbodiimide

    International Nuclear Information System (INIS)

    Sussman, M.R.; Slayman, C.W.

    1983-01-01

    The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 0 C is approximately 1000 M -1 min -1 , similar to values reported for the DCCD-binding proteolipid of F 0 -F 1 -type [H + ]-ATPases and for the sarcoplasmic reticulum [Ca +2 ]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [ 14 C]DCCD is incorporated into the M/sub r/ = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea the DCCD reacts with a single amino acid residue of the Neuspora [H + ]-ATPase, thereby blcoking ATP hydrolysis and proton translocation. 21 references, 5 figures, 2 tables

  7. Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy.

    Science.gov (United States)

    Spugnini, Enrico P; Citro, Gennaro; Fais, Stefano

    2010-05-08

    The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

  8. Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy

    Directory of Open Access Journals (Sweden)

    Fais Stefano

    2010-05-01

    Full Text Available Abstract The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

  9. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    Science.gov (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  10. Na+-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na+ into proteoliposomes via Na+ uniport mechanism

    Directory of Open Access Journals (Sweden)

    Soontharapirakkul Kanteera

    2010-08-01

    Full Text Available Abstract Background When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na+ from cells to maintain the cytoplasmic Na+ concentration. Until now, the ATPase involved in Na+ transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na+ transport of alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity conditions. Results The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+ but not K+, Li+ and Ca2+. The apparent Km values for Na+ and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F1F0-ATPase based on the usual subunit pattern and the protection against N,N'-dicyclohexylcarbodiimide inhibition of ATPase activity by Na+ in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na+ upon the addition of ATP. The apparent Km values for this uptake were 3.3 and 0.5 mM for Na+ and ATP, respectively. The mechanism of Na+ transport mediated by Na+-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na+-stimulated ATPase was observed upon the addition of ATP with Na+ but not with K+, Li+ and Ca2+. The Na+- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na+ uptake activity. The uptake of Na+ was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na+-stimulated ATPase of the proteoliposomes. Conclusion The data demonstrate that Na+-stimulated ATPase from A. halophytica, a likely member of F-type ATPase, functions as an electrogenic Na

  11. Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle

    DEFF Research Database (Denmark)

    Galuska, Dana; Kotova, Olga; Barres, Romain

    2009-01-01

    Skeletal muscle Na(+)-K(+)-ATPase plays a central role in the clearance of K(+) from the extracellular fluid, therefore maintaining blood [K(+)]. Na(+)-K(+)-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise......(+)-K(+)-ATPase activity after 4 wk of HFD. Exercise training restored alpha(1)-, alpha(2)-, and beta(1)-subunit expression and Na(+)-K(+)-ATPase activity to control levels and reduced beta(2)-subunit expression 2.2-fold (P ... phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the alpha(2)-subunit to plasma membrane was impaired by HFD, whereas alpha(1)-subunit translocation remained unchanged. Alterations in sodium pump...

  12. Towards the structure of yeast and mammalian P4-ATPases

    DEFF Research Database (Denmark)

    Lyons, Joseph; Laban, Milena; Mikkelsen, Stine

    2017-01-01

    P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic and bioche......P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic...... a basis for the analysis of reported mutagenesis data, we aim to solve the first molecular structures of the PS transporting P4-ATPases using electron microscopy. To date, negative stain EM analysis, on detergent, amphipol and saposin-lipoprotein nanoparticle (Salipro) reconstituted of both Drs2p/CDC50p...... and bATP8A2/CDC50A, has yielded comparable low-resolution envelopes of these two transporters, highlighting the bulk architecture of the complex. Current efforts and progress on the functional characterization and cryo-EM analysis of both lipid transporters reconstituted in Salipro are described...

  13. In and out of the cation pumps: P-type ATPase structure revisited

    DEFF Research Database (Denmark)

    Bublitz, Maike; Poulsen, Hanne; Morth, Jens Preben

    2010-01-01

    Active transport across membranes is a crucial requirement for life. P-type ATPases build up electrochemical gradients at the expense of ATP by forming and splitting a covalent phosphoenzyme intermediate, coupled to conformational changes in the transmembrane section where the ions are translocated....... The marked increment during the last three years in the number of crystal structures of P-type ATPases has greatly improved our understanding of the similarities and differences of pumps with different ion specificities, since the structures of the Ca2+-ATPase, the Na+,K+-ATPase and the H+-ATPase can now...

  14. V-ATPase as an effective therapeutic target for sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Perut, Francesca, E-mail: francesca.perut@ior.it [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)

    2014-01-01

    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

  15. Production and characterization of a monoclonal antibody to H+ATPase

    International Nuclear Information System (INIS)

    Yurko, M.; Fitch, F; Gluck, S.

    1986-01-01

    Acidification of endocytic vesicles is carried out by an ATP-dependent proton pump, H+ATPase, an FOF1 type enzyme comprised of at least 5 major subunits of 70, 56, 45, 35, and 17 kDa. A monoclonal antibody, H6.1, to H+ATPase from bovine kidney medulla, was raised to enable the structural characterization and localization of the pump. Several criteria were used to show that H6.1 recognized H+ATPase. 1.) H6.1 immunoprecipitated N-ethylmaleimide-sensitive and vanadate- and azide-insensitive solubilized ATPase activity (and GTPase activity) from both crude and purified enzyme preparations. 2.) H6.1 immunoprecipitated oligomycin-insensitive ATP-dependent proton transporting vesicles made from bovine kidney medulla, rat kidney, and CHO cells. 3.) H6.1 specifically immuno-precipitated the 5 subunits of H+ATPase from a partially purified preparation of the enzyme that had been labelled with I-125. H6.1 was then used as an immunocytochemical probe for the localization of H+ATPase. In bovine kidney medullary collecting duct, there was an intense apical staining of selected cells. In proximal tubule and in cultured CHO cells there was a granular pattern of staining characteristic of endocytic vesicles and lysosomes, suggesting that the kidney and CHO cell proton pumps are structurally related

  16. Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation.

    Science.gov (United States)

    Zhu, Shaotong; Vik, Steven B

    2015-08-21

    Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Modulation of proton pumping across proteoliposome membranes reconstituted with tonoplast H(+)-ATPase from cultured rice (Oryza sativa L. var. Boro) cells by acyl steryl glucoside and steryl glucoside.

    Science.gov (United States)

    Yamaguchi, Mineo; Kasamo, Kunihiro

    2002-07-01

    Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast.

  18. Crystal structure of the plasma membrane proton pump

    DEFF Research Database (Denmark)

    Pedersen, Bjørn P.; Buch-Pedersen, Morten Jeppe; Morth, J. Preben

    2007-01-01

    A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H1-ATPase (the proton pump) in plants and fungi1......-3, and Na1,K1-ATPase (the sodium-potassium pump) in animals4. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis5.The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na1,K1-ATPase and Ca21......- ATPase are type II6. Electron microscopy has revealed the overall shape of proton pumps7, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define...

  19. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    International Nuclear Information System (INIS)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H + translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10 5 , 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ ≅ 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F 0 F 1 ATPases

  20. Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

    DEFF Research Database (Denmark)

    Gantzel, Rasmus H; Mogensen, Louise S; Mikkelsen, Stine A

    2017-01-01

    Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the "giant...

  1. Tetrapropylammonium ion inhibits Na, K-ATPase by blocking a K+-translocation step

    International Nuclear Information System (INIS)

    Forbush, B. III

    1986-01-01

    Tetrapropylammonium ion (TPA) has previously been shown to inhibit the Na/K pump in human red cells in competition with K + at extracellular sites. The author investigated the action of TPA on the release of 86 Rb from the occluded state of Na, K-ATPase from dog kidney. Like K + and Rb + , TPA is found to prevent the release of one (''s'' for slower) of two tightly bound 86 Rb ions when the enzyme is phosphorylated from P/sub i/; the data are consistent with TPA binding to the K + ''f'' (faster) site with an affinity of ∼5mM. When 86 Rb is bound to the ''s'' site and unlabelled Rb + or K + to the ''f'' site by an appropriate sequence of additions, the 86 Rb can be released by addition of ATP to bring about the E 2 -E]3! conformational change and translocation. In contrast when TPA is bound to the ''f'' site there is no release of 86 Rb from the ''s'' site when ATP is added, suggesting that the conformational change is prevented by the bulky TPA molecule at the transport site. Addition of Na + (without nucleotide or P/sub i/) brings about an extremely rapid (>100s -1 ) release of 86 Rb when TPA is bound. The reason for this is not yet clear, but the effect dramatically illustrates simultaneous occupancy of binding sites by K + , TPA, and Na +

  2. Plasma Membrane H(+)-ATPase Regulation in the Center of Plant Physiology.

    Science.gov (United States)

    Falhof, Janus; Pedersen, Jesper Torbøl; Fuglsang, Anja Thoe; Palmgren, Michael

    2016-03-07

    The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  3. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.

    Science.gov (United States)

    Levin, Mikhail K; Gurjar, Madhura; Patel, Smita S

    2005-05-01

    Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

  4. Glutamate transporter activity promotes enhanced Na+/K+-ATPase -mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian R; Holm, Rikke; Vilsen, Bente

    2016-01-01

    , in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether...... the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter......+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i...

  5. Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

    2015-07-03

    In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.

  6. Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C.

    2015-01-01

    In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection

  7. Towards structural and functional analysis of the plant plasma membrane proton pump

    DEFF Research Database (Denmark)

    Justesen, Bo Højen

    The plasma membrane H+-ATPase is a proton pump essential for several physiological important processes in plants. Through the extrusion of protons from the cell, the PM H+-ATPase establishes and maintains a proton gradient used by proton coupled transporters and secondary active transport...... of nutrients and metabolites across the plasma membrane. Additional processes involving the PM H+-ATPase includes plant growth, development, and response to biotic and abiotic stresses. Extensive efforts have been made in attempts to elucidate the detailed physiological role and biochemical characteristics...... of plasma membrane H+-ATPases. Studies on the plasma membrane H+-ATPases have involved both in vivo and in vitro approaches, with the latter employing either solubilisation by detergent micelles, or reconstitution into lipid vesicles. Despite resulting in a large body of information on structure, function...

  8. In and out of the cation pumps: P-type ATPase structure revisited

    DEFF Research Database (Denmark)

    Bublitz, Maike; Poulsen, Hanne; Morth, Jens Preben

    2010-01-01

    . The marked increment during the last three years in the number of crystal structures of P-type ATPases has greatly improved our understanding of the similarities and differences of pumps with different ion specificities, since the structures of the Ca2+-ATPase, the Na+,K+-ATPase and the H+-ATPase can now......Active transport across membranes is a crucial requirement for life. P-type ATPases build up electrochemical gradients at the expense of ATP by forming and splitting a covalent phosphoenzyme intermediate, coupled to conformational changes in the transmembrane section where the ions are translocated...... be compared directly. Mechanisms for ion gating, charge neutralization and backflow prevention are starting to emerge from comparative structural analysis; and in combination with functional studies of mutated pumps this provides a framework for speculating on how the ions are bound and released as well...

  9. Vacuolar-proton-ATPase is involved in the response of cancer cells to ionizing radiation and is a new target for radiosensitization

    International Nuclear Information System (INIS)

    Hollister, T.C.; Paglin, S.; McMahill, M.S.; Gilles, F.; Yahalom, J.

    2001-01-01

    Purpose: We have recently described a novel response of human cancer cells to radiation consisting of accumulation of acidic vesicular organelles (AVO) (Cancer Research, 61:439-444, 2001). Acidification of AVO was inhibited by bafilomycin A1 - a specific inhibitor of vacuolar-proton-ATPase. To examine the role of AVO function in the cellular response to radiation, we determined the effect of specific inhibitors of v-H+-ATPase (bafilomycin and concanamycin) on the clonogenic survival of various cancer cell lines. In addition, Northern analysis of mRNA encoding v-H+-ATPase was performed to elucidate possible mechanisms of post-radiation v-H+-ATPase regulation. Materials and Methods: Total RNA was isolated from human cancer cell lines including MCF-7 (breast), LoVo (colon), and LNCaP (prostate) cells 48 hours post-irradiation, and Northern analyses were performed. RNA was separated on a denaturing formaldehyde gel and blotted to a nylon membrane. Probes made from cDNAs to v-H+-ATPase were radioactively labeled, hybridized to the membranes, and exposed to X-ray film for determination of mRNA expression levels. Clonogenic survival assays were performed with MCF-7 and LoVo cell lines. Cells were plated at 30 cells/cm 2 , and irradiated 24 hours post-plating. Bafilomycin or concanamycin were added to culture media in various concentrations at the time of irradiation. The media was changed after a 24-hour incubation, and colonies were stained with crystal violet for counting 7 days post-irradiation. Results: Radiation-induced accumulation of acidic vesicular organelles in MCF-7, LoVo and LNCaP cells was associated with a two-fold increase in the steady-state level of mRNA for subunit c of v-H+-ATPase. Bafilomycin and concanamycin increased clonogenic cell death after irradiation in a dose-dependent manner. At low concentrations (bafilomycin 2nM, concanamycin 2pM) these agents acted as radiosensitizers, without significant toxicity to unirradiated cells. At 2nM, bafilomycin

  10. Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

    Science.gov (United States)

    Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

    2018-05-22

    It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging.

    Science.gov (United States)

    Schwartz, Chad; Fang, Huaming; Huang, Lisa; Guo, Peixuan

    2012-03-01

    Many cells and double-stranded DNA (dsDNA) viruses contain an AAA(+) ATPase that assembles into oligomers, often hexamers, with a central channel. The dsDNA packaging motor of bacteriophage phi29 also contains an ATPase to translocate dsDNA through a dodecameric channel. The motor ATPase has been investigated substantially in the context of the entire procapsid. Here, we report the sequential action between the ATPase and additional motor components. It is suggested that the contact of ATPase to ATP resulted in its conformational change to a higher binding affinity toward dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action that is speculated to push dsDNA to pass the connector channel. Our results suggest that dsDNA packaging goes through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Many packaging models have previously been proposed, and the packaging mechanism has been contingent upon the number of nucleotides packaged per ATP relative to the 10.5 bp per helical turn for B-type dsDNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five or six discrete steps of dsDNA translocation. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP.

  12. Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.

    Science.gov (United States)

    Oliveira, Leonor; Cuervo, Ana; Tavares, Paulo

    2010-03-05

    DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

  13. Specialized functional diversity and interactions of the Na,K-ATPase

    Directory of Open Access Journals (Sweden)

    Igor I. Krivoi

    2016-05-01

    Full Text Available Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions and protein kinase signaling pathways. In addition to its ‘classical’ function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function.

  14. Respiration-dependent proton translocation in alkalophilic Bacillus firmus RAB and its non-alkalophilic mutant derivative.

    Science.gov (United States)

    Lewis, R J; Krulwich, T A; Reynafarje, B; Lehninger, A L

    1983-02-25

    Obligately alkalophilic Bacillus firmus RAB had a higher molar growth yield on L-malate (Ymal = 38 mg, dry weight/mmol of L-malate) than its non-alkalophilic mutant derivative, strain RABN (Ymal = 12 mg, dry weight/mmol of L-malate). Measurements of respiration dependent proton translocation by the two strains in the presence of K+ and valinomycin showed that the alkalophile also has much higher H+/O stoichiometries (at pH 9.0) than does the mutant (at pH 7.0). H+/O ratios for B. firmus RAB at pH 9.0 were as high as 13, with a frequently observed value of 9. These high values were observed in the first phase of a set of biphasic curves for both oxygen consumption and proton ejection. At pH 7.0, both the wild type and the mutant exhibited H+/O ratios near 4 in a single phase of oxygen consumption and proton ejection. The results are consistent with suggestions that the alkalophilic respiratory chain is especially well adapted for effective energy transduction at alkaline but not neutral pH.

  15. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

    of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...

  16. Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea.

    Science.gov (United States)

    Rzechorzek, Neil J; Blackwood, John K; Bray, Sian M; Maman, Joseph D; Pellegrini, Luca; Robinson, Nicholas P

    2014-11-25

    The HerA ATPase cooperates with the NurA nuclease and the Mre11-Rad50 complex for the repair of double-strand DNA breaks in thermophilic archaea. Here we extend our structural knowledge of this minimal end-resection apparatus by presenting the first crystal structure of hexameric HerA. The full-length structure visualizes at atomic resolution the N-terminal HerA-ATP synthase domain and a conserved C-terminal extension, which acts as a physical brace between adjacent protomers. The brace also interacts in trans with nucleotide-binding residues of the neighbouring subunit. Our observations support a model in which the coaxial interaction of the HerA ring with the toroidal NurA dimer generates a continuous channel traversing the complex. HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion. This system differs substantially from the bacterial end-resection paradigms. Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.

  17. MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

    Science.gov (United States)

    Sharma, Stuti; Oot, Rebecca A; Wilkens, Stephan

    2018-05-12

    Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary motor proton pumps that acidify intracellular compartments and in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

    Science.gov (United States)

    Schulz, Sarah; Wilkes, Martin; Mills, Deryck J; Kühlbrandt, Werner; Meier, Thomas

    2017-04-01

    The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N 1 N o -type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c 17 ring is H + specific, demonstrating that the ATPase is proton-coupled. The c 17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  19. Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

    DEFF Research Database (Denmark)

    Hartmann-Petersen, R; Tanaka, K; Hendil, K B

    2001-01-01

    and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross......-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7......, these data establish the relative orientations of ATPases with respect to the 20S proteasome....

  20. Active glucose transport and proton pumping in tonoplast membrane of Zea mays L. coleoptiles are inhibited by anti-H+-ATPase antibodies

    International Nuclear Information System (INIS)

    Rausch, T.; Butcher, D.N.; Taiz, L.

    1987-01-01

    A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg 2+ -ATP dependent accumulation of 3-O-methyl-D-glucose (OMG) as determined by a membrane filtration technique using 14 C-labeled substrate. OMG transport showed an apparently saturable component with a K/sub m/ of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H + -ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes

  1. Structure and mechanism of the ATPase that powers viral genome packaging.

    Science.gov (United States)

    Hilbert, Brendan J; Hayes, Janelle A; Stone, Nicholas P; Duffy, Caroline M; Sankaran, Banumathi; Kelch, Brian A

    2015-07-21

    Many viruses package their genomes into procapsids using an ATPase machine that is among the most powerful known biological motors. However, how this motor couples ATP hydrolysis to DNA translocation is still unknown. Here, we introduce a model system with unique properties for studying motor structure and mechanism. We describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide-dependent conformational changes involving a large rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in a neighboring motor subunit, illustrating that previous models for motor structure need revision. Our findings allow us to derive a structural model for the motor ring, which we validate using small-angle X-ray scattering and comparisons with previously published data. We illustrate the model's predictive power by identifying the motor's DNA-binding and assembly motifs. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.

  2. Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

    Science.gov (United States)

    Majumdar, Arkajo; Kar, Rup Kumar

    2018-07-01

    Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

  3. Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

    Science.gov (United States)

    Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

    2015-12-01

    The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

  4. Structure of V-type ATPase from Clostridium fervidus by electron microscopy

    NARCIS (Netherlands)

    Boekema, EJ; Ubbink-Kok, T; Lolkema, JS; Brisson, A; Konings, WN

    F-type and V-type ATPases couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes and have similarities in structure and mechanism. In both types of enzymes three main parts can be distinguished: headpiece, membrane-bound piece and stalk region. We report

  5. Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation.

    NARCIS (Netherlands)

    Koenderink, J.B.; Zifarelli, G.; Qiu, L.Y.; Schwarz, W.; Pont, J.J.H.H.M. de; Bamberg, E.; Friedrich, T.

    2005-01-01

    The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms).

  6. Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

    Science.gov (United States)

    Vedovato, Natascia

    2014-01-01

    A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified. PMID

  7. Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps.

    Science.gov (United States)

    Vedovato, Natascia; Gadsby, David C

    2014-04-01

    A single Na(+)/K(+)-ATPase pumps three Na(+) outwards and two K(+) inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na(+) than K(+) generates outward current across the cell membrane. Less well understood is the ability of Na(+)/K(+) pumps to generate an inward current of protons. Originally noted in pumps deprived of external K(+) and Na(+) ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K(+) and Na(+) concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na(+) release from phosphorylated Na(+)/K(+) pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na(+)/K(+) pumps that enables proton import is not required for completion of the 3 Na(+)/2 K(+) transport cycle. However, the back-step occurs readily during Na(+)/K(+) transport when external K(+) ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na(+)-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na(+) and K(+) ions that passes through binding site II. The inferred occurrence of Na(+)/K(+) exchange and H(+) import during the same conformational cycle of a single molecule identifies the Na(+)/K(+) pump as a hybrid transporter. Whether Na(+)/K(+) pump-mediated proton inflow may have any physiological or

  8. Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

    Science.gov (United States)

    Graham, Laurie A; Finnigan, Gregory C; Kane, Patricia M

    2018-06-01

    Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma - phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Palmgren, Michael Gjedde; Buch-Pedersen, Morten Jeppe

    The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic...

  10. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  11. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    International Nuclear Information System (INIS)

    Kohio, Hinissan P.; Adamson, Amy L.

    2013-01-01

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

  12. Regulation of vacuolar H+-ATPase in microglia by RANKL

    International Nuclear Information System (INIS)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

    2009-01-01

    Vacuolar H + -ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  13. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    Science.gov (United States)

    Monroe, Nicole; Hill, Christopher P

    2016-05-08

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Characterization and effect of light on the plasma membrane H(+) -ATPase of bean leaves

    Science.gov (United States)

    Linnemeyer, P. A.; Van Volkenburgh, E.; Cleland, R. E.

    1990-01-01

    Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the Km was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, Km, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.

  15. Protein-protein interactions within the ensemble, eukaryotic V-ATPase, and its concerted interactions with cellular machineries.

    Science.gov (United States)

    Balakrishna, Asha Manikkoth; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

    2015-10-01

    The V1VO-ATPase (V-ATPase) is the important proton-pump in eukaryotic cells, responsible for pH-homeostasis, pH-sensing and amino acid sensing, and therefore essential for cell growths and metabolism. ATP-cleavage in the catalytic A3B3-hexamer of V1 has to be communicated via several so-called central and peripheral stalk units to the proton-pumping VO-part, which is membrane-embedded. A unique feature of V1VO-ATPase regulation is its reversible disassembly of the V1 and VO domain. Actin provides a network to hold the V1 in proximity to the VO, enabling effective V1VO-assembly to occur. Besides binding to actin, the 14-subunit V-ATPase interacts with multi-subunit machineries to form cellular sensors, which regulate the pH in cellular compartments or amino acid signaling in lysosomes. Here we describe a variety of subunit-subunit interactions within the V-ATPase enzyme during catalysis and its protein-protein assembling with key cellular machineries, essential for cellular function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.

    2015-01-01

    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

  17. Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

    Science.gov (United States)

    Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

    1999-07-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

  18. Targeting vacuolar H+-ATPases as a new strategy against cancer.

    Science.gov (United States)

    Fais, Stefano; De Milito, Angelo; You, Haiyan; Qin, Wenxin

    2007-11-15

    Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

  19. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  20. Protonated form: the potent form of potassium-competitive acid blockers.

    Directory of Open Access Journals (Sweden)

    Hua-Jun Luo

    Full Text Available Potassium-competitive acid blockers (P-CABs are highly safe and active drugs targeting H+,K+-ATPase to cure acid-related gastric diseases. In this study, we for the first time investigate the interaction mechanism between the protonated form of P-CABs and human H+,K+-ATPase using homology modeling, molecular docking, molecular dynamics and binding free energy calculation methods. The results explain why P-CABs have higher activities with higher pKa values or at lower pH. With positive charge, the protonated forms of P-CABs have more competitive advantage to block potassium ion into luminal channel and to bind with H+,K+-ATPase via electrostatic interactions. The binding affinity of the protonated form is more favorable than that of the neutral P-CABs. In particular, Asp139 should be a very important binding site for the protonated form of P-CABs through hydrogen bonds and electrostatic interactions. These findings could promote the rational design of novel P-CABs.

  1. Protonated form: the potent form of potassium-competitive acid blockers.

    Science.gov (United States)

    Luo, Hua-Jun; Deng, Wei-Qiao; Zou, Kun

    2014-01-01

    Potassium-competitive acid blockers (P-CABs) are highly safe and active drugs targeting H+,K+-ATPase to cure acid-related gastric diseases. In this study, we for the first time investigate the interaction mechanism between the protonated form of P-CABs and human H+,K+-ATPase using homology modeling, molecular docking, molecular dynamics and binding free energy calculation methods. The results explain why P-CABs have higher activities with higher pKa values or at lower pH. With positive charge, the protonated forms of P-CABs have more competitive advantage to block potassium ion into luminal channel and to bind with H+,K+-ATPase via electrostatic interactions. The binding affinity of the protonated form is more favorable than that of the neutral P-CABs. In particular, Asp139 should be a very important binding site for the protonated form of P-CABs through hydrogen bonds and electrostatic interactions. These findings could promote the rational design of novel P-CABs.

  2. Bioinformatics Study of m.9053G>A Mutation at the Gene in Relation to Type 2 Diabetes Mellitus and Cataract Diseases

    Directory of Open Access Journals (Sweden)

    *Iman Permana Maksum

    2017-09-01

    Full Text Available The mitochondrial disease often associated with various illnesses in relation to the activity of cells metabolites and the synthesis of adenosine triphosphate (ATP, including alteration in the mitochondrial DNA. The mutation of m.9053G>A at the ATP6 gene was found in patients with type 2 diabetes mellitus (DM type 2 and cataract. Therefore, this mutation is predicted to be clinical features of the 2 diseases. ATP6 gene encodes protein subunit of ATPase6, a part of ATP synthase, which is important in the electron transfer and proton translocation in intracellular respiration system. This study aims to investigate the mutation effect of m.9053G>A at the ATP6 gene (S167N to the structure and function of ATPase6 using bioinformatics method. The structure of ATPase6 was constructed using homology modeling method. The crystal structure of bovine’s ATP synthase (Protein Data Bank ID 5FIL was used as a template because of high sequence similarity (77% and coverage (96% of the input sequence. The effect of mutation was investigated at the proton translocation channel of ATPase6. It is predicted that the channel was disrupted due to changes in electrostatic potential from serine to asparagine. Furthermore, molecular docking suggests that water binding on the proton translocation channel in the S167N mutant was different from the wild type. The result of this study is hoped to be useful in the development of a new genetic marker for DM type 2 and cataract.

  3. A non-equilibrium thermodynamics model of reconstituted Ca(2+)-ATPase.

    Science.gov (United States)

    Waldeck, A R; van Dam, K; Berden, J; Kuchel, P W

    1998-01-01

    A non-equilibrium thermodynamics (NET) model describing the action of completely coupled or 'slipping' reconstituted Ca(2+)-ATPase is presented. Variation of the coupling stoichiometries with the magnitude of the electrochemical gradients, as the ATPase hydrolyzes ATP, is an indication of molecular slip. However, the Ca2+ and H+ membrane-leak conductances may also be a function of their respective gradients. Such non-ohmic leak typically yields 'flow-force' relationships that are similar to those that are obtained when the pump slips; hence, caution needs to be exercised when interpreting data of Ca(2+)-ATPase-mediated fluxes that display a non-linear dependence on the electrochemical proton (delta mu H) and/or calcium gradients (delta mu Ca). To address this issue, three experimentally verifiable relationships differentiating between membrane leak and enzymic slip were derived. First, by measuring delta mu H as a function of the rate of ATP hydrolysis by the enzyme. Second, by measuring the overall 'efficiency' of the pump as a function of delta mu H. Third, by measuring the proton ejection rate by the pump as a function of its ATP hydrolysis rate.

  4. Torque-coupled thermodynamic model for FoF1 -ATPase

    Science.gov (United States)

    Ai, Guangkuo; Liu, Pengfei; Ge, Hao

    2017-05-01

    FoF1 -ATPase is a motor protein complex that utilizes transmembrane ion flow to drive the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (Pi). While many theoretical models have been proposed to account for its rotary activity, most of them focus on the Fo or F1 portions separately rather than the complex as a whole. Here, we propose a simple but new torque-coupled thermodynamic model of FoF1 -ATPase. Solving this model at steady state, we find that the monotonic variation of each portion's efficiency becomes much more robust over a wide range of parameters when the Fo and F1 portions are coupled together, as compared to cases when they are considered separately. Furthermore, the coupled model predicts the dependence of each portion's kinetic behavior on the parameters of the other. Specifically, the power and efficiency of the F1 portion are quite sensitive to the proton gradient across the membrane, while those of the Fo portion as well as the related Michaelis constants for proton concentrations respond insensitively to concentration changes in the reactants of ATP synthesis. The physiological proton gradient across the membrane in the Fo portion is also shown to be optimal for the Michaelis constants of ADP and phosphate in the F1 portion during ATP synthesis. Together, our coupled model is able to predict key dynamic and thermodynamic features of the FoF1 -ATPase in vivo semiquantitatively, and suggests that such coupling approach could be further applied to other biophysical systems.

  5. ISOLATION AND CHARACTERIZATION OF THE HIGH-AFFINITY K+-TRANSLOCATING ATPASE FROM RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    ABEE, T; SIEBERS, A; ALTENDORF, K; KONINGS, WN

    1992-01-01

    Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K+-stimulated and Mg2+-stimulated ATPase was purified from membranes of these cells by solubilization with

  6. Characterization of the vacuolar H sup + -ATPase of higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Manolson, M F

    1988-01-01

    The tonoplast H{sup +}-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides. Kinetic analysis of ({alpha}-{sup 32}P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ({sup 14}C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H{sup +}-ATPase is a multimer composed of at least three polypeptides. Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H{sup +}-ATPase of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F{sub 0}F{sub 1} and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

  7. Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate

    International Nuclear Information System (INIS)

    Williams, L.E.; Schueler, S.B.; Briskin, D.P.

    1990-01-01

    The GTP-driven component of Ca 2+ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca 2+ -translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45 Ca 2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca 2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca 2+ -ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45 Ca 2+ -uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45 Ca 2+ uptake represents the capacity of the plasma membrane Ca 2+ -translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ- 32 P]GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca 2+ -ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca 2+ -ATPases present in animal cells

  8. Regulation of branchial V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias).

    Science.gov (United States)

    Tresguerres, Martin; Katoh, Fumi; Fenton, Heather; Jasinska, Edyta; Goss, Greg G

    2005-01-01

    To study the mechanisms of branchial acid-base regulation, Pacific spiny dogfish were infused intravenously for 24 h with either HCl (495+/- 79 micromol kg(-1) h(-1)) or NaHCO(3) (981+/-235 micromol kg(-1) h(-1)). Infusion of HCl produced a transient reduction in blood pH. Despite continued infusion of acid, pH returned to normal by 12 h. Infusion of NaHCO(3) resulted in a new steady-state acid-base status at approximately 0.3 pH units higher than the controls. Immunostained serial sections of gill revealed the presence of separate vacuolar proton ATPase (V-H(+)-ATPase)-rich or sodium-potassium ATPase (Na(+)/K(+)-ATPase)-rich cells in all fish examined. A minority of the cells also labeled positive for both transporters. Gill cell membranes prepared from NaHCO(3)-infused fish showed significant increases in both V-H(+)-ATPase abundance (300+/-81%) and activity. In addition, we found that V-H(+)-ATPase subcellular localization was mainly cytoplasmic in control and HCl-infused fish, while NaHCO(3)-infused fish demonstrated a distinctly basolateral staining pattern. Western analysis in gill membranes from HCl-infused fish also revealed increased abundance of Na(+)/H(+) exchanger 2 (213+/-5%) and Na(+)/K(+)-ATPase (315+/-88%) compared to the control.

  9. Proton pump inhibitors inhibit pancreatic secretion

    DEFF Research Database (Denmark)

    Wang, Jing; Barbuskaite, Dagne; Tozzi, Marco

    2015-01-01

    +/K+-ATPases are expressed and functional in human pancreatic ducts and whether proton pump inhibitors (PPIs) have effect on those. Here we show that the gastric HKα1 and HKβ subunits (ATP4A; ATP4B) and non-gastric HKα2 subunits (ATP12A) of H+/K+-ATPases are expressed in human pancreatic cells. Pumps have similar...... of major ions in secretion follow similar excretory curves in control and PPI treated animals. In addition to HCO3-, pancreas also secretes K+. In conclusion, this study calls for a revision of the basic model for HCO3- secretion. We propose that proton transport is driving secretion, and that in addition...

  10. Molecular determinants of nucleolar translocation of RNA helicase A

    International Nuclear Information System (INIS)

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-01-01

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

  11. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  12. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  13. Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

    1999-01-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

  14. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

  15. Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide

    International Nuclear Information System (INIS)

    Arai, H.; Berne, M.; Forgac, M.

    1987-01-01

    N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [ 14 C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump

  16. A voltage-gated pore for translocation of tRNA

    Energy Technology Data Exchange (ETDEWEB)

    Koley, Sandip; Adhya, Samit, E-mail: nilugrandson@gmail.com

    2013-09-13

    Highlights: •A tRNA translocating complex was assembled from purified proteins. •The complex translocates tRNA at a membrane potential of ∼60 mV. •Translocation requires Cys and His residues in the Fe–S center of RIC6 subunit. -- Abstract: Very little is known about how nucleic acids are translocated across membranes. The multi-subunit RNA Import Complex (RIC) from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes, but the nature of the translocation pore remains unknown. We show that subunits RIC6 and RIC9 assemble on the membrane in presence of subunit RIC4A to form complex R3. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of ∼20 nm rim diameter and ∼1 nm depth. R3 induced translocation of tRNA into liposomes when the pH of the medium was lowered to ∼6 in the absence of ATP. R3-mediated tRNA translocation could also be induced at neutral pH by a K{sup +} diffusion potential with an optimum of 60–70 mV. Point mutations in the Cys{sub 2}–His{sub 2} Fe-binding motif of RIC6, which is homologous to the respiratory Complex III Fe–S protein, abrogated import induced by low pH but not by K{sup +} diffusion potential. These results indicate that the R3 complex forms a pore that is gated by a proton-generated membrane potential and that the Fe–S binding region of RIC6 has a role in proton translocation. The tRNA import complex of L. tropica thus contains a novel macromolecular channel distinct from the mitochondrial protein import pore that is apparently involved in tRNA import in some species.

  17. F F1-ATPase as biosensor to detect single virus

    International Nuclear Information System (INIS)

    Liu, XiaoLong; Zhang, Yun; Yue, JiaChang; Jiang, PeiDong; Zhang, ZhenXi

    2006-01-01

    F F 1 -ATPase within chromatophore was constructed as a biosensor (immuno-rotary biosensor) for the purpose of capturing single virus. Capture of virus was based on antibody-antigen reaction. The detection of virus based on proton flux change driven by ATP-synthesis of F F 1 -ATPase, which was indicated by F1300, was directly observed by a fluorescence microscope. The results demonstrate that the biosensor loading of virus particles has remarkable signal-to-noise ratio (3.8:1) compared to its control at single molecular level, and will be convenient, quick, and even super-sensitive for detecting virus particles

  18. Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.

    Science.gov (United States)

    Morales-Rios, Edgar; Watt, Ian N; Zhang, Qifeng; Ding, Shujing; Fearnley, Ian M; Montgomery, Martin G; Wakelam, Michael J O; Walker, John E

    2015-09-01

    The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex. © 2015 The Authors.

  19. Decoding P4-ATPase substrate interactions.

    Science.gov (United States)

    Roland, Bartholomew P; Graham, Todd R

    Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca 2+ , a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.

  20. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  1. PGE2 suppresses intestinal T cell function in thermal injury: a cause of enhanced bacterial translocation.

    Science.gov (United States)

    Choudhry, M A; Fazal, N; Namak, S Y; Haque, F; Ravindranath, T; Sayeed, M M

    2001-09-01

    Increased gut bacterial translocation in burn and trauma patients has been demonstrated in a number of previous studies, however, the mechanism for such an increased gut bacterial translocation in injured patients remains poorly understood. Utilizing a rat model of burn injury, in the present study we examined the role of intestinal immune defense by analyzing the T cell functions. We investigated if intestinal T cells dysfunction contributes to bacterial translocation after burn injury. Also our study determined if burn-mediated alterations in intestinal T cell functions are related to enhanced release of PGE2. Finally, we examined whether or not burn-related alterations in intestinal T cell function are due to inappropriate activation of signaling molecule P59fyn, which is required for T cell activation and proliferation. The results presented here showed an increase in gut bacterial accumulation in mesenteric lymph nodes after thermal injury. This was accompanied by a decrease in the intestinal T cell proliferative responses. Furthermore, the treatments of burn-injured animals with PGE2 synthesis blocker (indomethacin or NS398) prevented both the decrease in intestinal T cell proliferation and enhanced bacterial translocation. Finally, our data suggested that the inhibition of intestinal T cell proliferation could result via PGE2-mediated down-regulation of the T cell activation-signaling molecule P59fyn. These findings support a role of T cell-mediated immune defense against bacterial translocation in burn injury.

  2. Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses.

    Science.gov (United States)

    Janicka, Małgorzata; Wdowikowska, Anna; Kłobus, Grażyna

    2018-01-01

    Plasma membrane (PM) H + -ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H + -ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

  3. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.

    2007-01-01

    Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report...... that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel...

  4. Novel target design for enhanced laser driven proton acceleration

    Directory of Open Access Journals (Sweden)

    Malay Dalui

    2017-09-01

    Full Text Available We demonstrate a simple method of preparing structured target for enhanced laser-driven proton acceleration under target-normal-sheath-acceleration scheme. A few layers of genetically modified, clinically grown micron sized E. Coli bacteria cell coated on a thin metal foil has resulted in an increase in the maximum proton energy by about 1.5 times and the total proton yield is enhanced by approximately 25 times compared to an unstructured reference foil at a laser intensity of 1019 W/cm2. Particle-in-cell simulations on the system shows that the structures on the target-foil facilitates anharmonic resonance, contributing to enhanced hot electron production which leads to stronger accelerating field. The effect is observed to grow as the number of structures is increased in the focal area of the laser pulse.

  5. Effect of TGFβ on Na+/K+ ATPase activity in megakaryocytes

    International Nuclear Information System (INIS)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina; Laufer, Stefan; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

    2014-01-01

    Highlights: • TGFß1 markedly up-regulates Na + /K + ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na + /K + ATPase generates the Na + and K + concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na + /K + ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na + /K + ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na + /K + ATPase activity determined from K + induced current utilizing whole cell patch clamp. The pump current (I pump ) was determined in the absence and presence of Na + /K + ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I pump was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion, TGFß1 is a powerful regulator of megakaryocytic Na + /K + ATPase activity

  6. Radiation inactivation analysis of chloroplast CF0-CF1 ATPase

    International Nuclear Information System (INIS)

    Wang, M.Y.; Chien, L.F.; Pan, R.L.

    1988-01-01

    Radiation inactivation technique was employed to measure the functional size of adenosine triphosphatase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 450 +/- 24 kilodaltons; for phenazine methosulfate-mediated ATP synthesis, 613 +/- 33 kilodaltons; and for methanol-activated ATP hydrolysis, 280 +/- 14 kilodaltons. The difference (170 +/- 57 kilodaltons) between 450 +/- 24 and 280 +/- 14 kilodaltons is explained to be the molecular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, II, and III of coupling factor 0 is 1:2:15. Ca2+- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as SO 3 (2-) and CO 3 (2-) increased the molecular mass for both ATPase's (except trypsin-activated Mg2+-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation

  7. Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

    Science.gov (United States)

    Scherer, G F

    1984-03-01

    A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

  8. Partial purification and properties of adenosine triphosphatase (ATPase) from liver fluke Fasciola hepatica.

    Science.gov (United States)

    Hassan, Husain; Abeer, Ali

    2014-01-01

    The adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3.;ATPase) is a membrane -bound enzyme which transport protons across the plasma membrane using ATP as an energy source. The adenosine triphosphatase (ATPase ; EC: 3.6.1.3) was extracted from membrane preparations of adult Fasciola hepatica by chloroform treatment and purified by means of ammonium sulphate fractionation, gel filtration on sephadex G-200 and DEAE- Cellulose chromatography. The molecular weight was calculated to be 305.000 dalton by gel filtration. Kinetic experiments demonstrated a biphasic linear lineweaver - burk relationship (km=0.142 and 1.66 mM) thus revealing the existence of two substrate binding enzyme sites. In our study revealed that partial inhibition of Mg²⁺ dependent purified enzyme by oligomycin suggest the absence of mitochondrial ATPase in F. hepatica.

  9. ULF waves associated with enhanced subauroral proton precipitation

    Science.gov (United States)

    Immel, Thomas J.; Mende, S. B.; Frey, H. U.; Patel, J.; Bonnell, J. W.; Engebretson, M. J.; Fuselier, S. A.

    Several types of sub-auroral proton precipitation events have been identified using the Spectrographic Imager (SI) onboard the NASA-IMAGE satellite, including dayside subauroral proton flashes and detached proton arcs in the dusk sector. These have been observed at various levels of geomagnetic activity and solar wind conditions and the mechanism driving the precipitation has often been assumed to be scattering of protons into the loss cone by enhancement of ion-cyclotron waves in the interaction of the thermal plasmaspheric populations and more energetic ring current particles. Indeed, recent investigation of the detached arcs using the MPA instruments aboard the LANL geosynchronous satellites has shown there are nearly always heightened densities of cold plasma on high-altitude field lines which map down directly to the sub-auroral precipitation. If the ion-cyclotron instability is a causative mechanism, the enhancement of wave activity at ion-cyclotron frequencies should be measurable. It is here reported that magnetic pulsations in the Pc1 range occur in the vicinity of each of 4 detached arcs observed in 2000-2002, though with widely varying signatures. Additionally, longer period pulsations in the Pc5 ranges are also observed in the vicinity of the arcs, leading to the conclusion that a bounce-resonance of ring-current protons with the azimuthal Pc5 wave structure may also contribute to the detached precipitation.

  10. Long-term regulation of Na,K-ATPase pump during T-cell proliferation.

    Science.gov (United States)

    Karitskaya, Inna; Aksenov, Nikolay; Vassilieva, Irina; Zenin, Valerii; Marakhova, Irina

    2010-09-01

    The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G(0)/G(1)/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [(3)H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase alpha1-subunit and beta1-subunit mRNAs and significant increase in the Na,K-ATPase alpha1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

  11. Uptake and translocation of [14C]-monoethanolamine in barley plants

    International Nuclear Information System (INIS)

    Eckert, H.; Bergmann, H.; Reissmann, P.

    1988-01-01

    Uptake and translocation of 14 C-monoethanolamine (EA) and its hydrochloride were investigated after application to an unwounded part of the fifth leaf from the main shoot of intact spring barley plants. After 48 and 72 h, resp., the free EA base was both absorbed rapidly and translocated out of the feeding leaf. The absorbed 14 C preferably migrated to the tillers, which resulted in an approximately uniform distribution of the radioactivity in the above ground parts of the plant after the uptake phase (similar 14 C concentrations in the main shoot and tillers), whereas only few radioactivity moved to the roots. On the other hand, the protonated EA (EA-HCl) exhibited both a reduced uptake and a restricted mobility. The bulk of radioactivity remained in the main shoot. As a consequence of the principally analogous metabolism of EA and its protonated form, the translocation differences are compensated during ontogenesis. When the plants reached maturity, similar distribution patterns could be found in which the kernels represented a considerable sink. (author)

  12. Enhancement of the incoherent scattering plasma lines due to precipitating protons and secondary electrons

    International Nuclear Information System (INIS)

    Bjoernaa, N.; Havnes, O.; Jensen, J.O.; Trulsen, J.

    1982-01-01

    Precipitating protons in the energy range 1-100 keV are regularly present in the auroral ionosphere. These protons will produce enhancements in the intensity of the upshifted plasma line of the incoherently scattered spectrum. Similarly, secondary electrons produced by the precipitating protons give rise to enhanced plasma line intensities. For a quantitative discussion of these effects an experimentally measured proton flux is adapted and the corresponding secondary electron flux calculated. These particle fluxes are then applied in connection with the EISCAT radar facility. Both fluxes give rise to enhancements of the order of 20. It is possible to separate between proton and electron contributions to the enhanced plasma lines for scattering heights above the source region of secondary electrons. (Auth.)

  13. Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

    Science.gov (United States)

    Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

    2014-01-01

    The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

  14. Plasmalemmal V-H+-ATPases regulate intracellular pH in human lung microvascular endothelial cells

    International Nuclear Information System (INIS)

    Rojas, Jose D.; Sennoune, Souad R.; Maiti, Debasish; Martinez, Gloria M.; Bakunts, Karina; Wesson, Donald E.; Martinez-Zaguilan, Raul

    2004-01-01

    The lung endothelium layer is exposed to continuous CO 2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na + /H + exchanger and HCO 3 - -dependent H + -transporting mechanisms regulate intracellular pH (pH cyt ) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H + -ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na + /H + exchanger and HCO 3 - -based H + -transporting mechanisms, to maintain pH cyt homeostasis. Immunocytochemical studies revealed V-H + -ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na + and HCO 3 - that were similar to those observed in the presence of either Na + , or Na + and HCO 3 - . The Na + - and HCO 3 - -independent pH cyt recovery was inhibited by bafilomycin A 1 , a V-H + -ATPase inhibitor. These studies show a Na + - and HCO 3 - -independent pH cyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases

  15. The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues Rosa Laura López-Marqués1, Lisbeth Rosager Poulsen1, Katharina Meffert2, Thomas Pomorski2, Michael Gjedde Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation...... physiological function.   1 Poulsen, L.R; López-Marqués, R.L et al. (2008) The Arabidopsis P4-ATPase ALA3 localizes to the Golgi and requires a ß-subunit to function in lipid translocation and secretory vesicle formation. The Plant Cell, vol. 20, 658-676. 2 Gomès, E. et al. (2000) Chilling tolerance...... in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases. The Plant Cell, vol. 12, 2441-2453....

  16. The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues

    DEFF Research Database (Denmark)

    Poulsen, Lisbeth Rosager

      The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues Rosa Laura López-Marqués1, Lisbeth Rosager Poulsen1, Katharina Meffert2, Thomas Pomorski2, Michael Gjedde Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation...... physiological function.   1 Poulsen, L.R; López-Marqués, R.L et al. (2008) The Arabidopsis P4-ATPase ALA3 localizes to the Golgi and requires a ß-subunit to function in lipid translocation and secretory vesicle formation. The Plant Cell, vol. 20, 658-676. 2 Gomès, E. et al. (2000) Chilling tolerance...... in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases. The Plant Cell, vol. 12, 2441-2453....

  17. Controllable Shrinking of Glass Capillary Nanopores Down to sub-10 nm by Wet-Chemical Silanization for Signal-Enhanced DNA Translocation.

    Science.gov (United States)

    Xu, Xiaolong; Li, Chuanping; Zhou, Ya; Jin, Yongdong

    2017-10-27

    Diameter is a major concern for nanopore based sensing. However, directly pulling glass capillary nanopore with diameter down to sub-10 nm is very difficult. So, post treatment is sometimes necessary. Herein, we demonstrate a facile and effective wet-chemical method to shrink the diameter of glass capillary nanopore from several tens of nanometers to sub-10 nm by disodium silicate hydrolysis. Its benefits for DNA translocation are investigated. The shrinking of glass capillary nanopore not only slows down DNA translocation, but also enhances DNA translocation signal and signal-to-noise ratio significantly (102.9 for 6.4 nm glass nanopore, superior than 15 for a 3 nm silicon nitride nanopore). It also affects DNA translocation behaviors, making the approach and glass capillary nanopore platform promising for DNA translocation studies.

  18. RNAi-directed downregulation of vacuolar H(+ -ATPase subunit a results in enhanced stomatal aperture and density in rice.

    Directory of Open Access Journals (Sweden)

    Huiying Zhang

    Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

  19. Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth.

    Science.gov (United States)

    Wang, Yin; Noguchi, Ko; Ono, Natsuko; Inoue, Shin-ichiro; Terashima, Ichiro; Kinoshita, Toshinori

    2014-01-07

    Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO2, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H(+)-ATPase, and plasma membrane inward-rectifying K(+) channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO2 uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H(+)-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with ∼42-63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were ∼36-41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K(+) channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H(+)-ATPase in guard cells is useful for the promotion of plant growth.

  20. Relative biological effectiveness of protons and heavy particles

    International Nuclear Information System (INIS)

    Vyglenov, A.; Fedorenko, B.; Kabachenko, A.

    1986-01-01

    The genetic effectiveness was studied of protons (9 GeB/nuclon, 0,72 Gy/min), α-particles (4 GeB/nuclon, 0,9 Gy/min) and carbon ions (4 GeB/nuclon 0,36 Gy/min). The translocation yield in mouse spermatogonia was used as indicator of radiation-induced genetic injury. Reciprocal translocation were registered six months after the irradiation on spermatocytes in diakinesmetaphase I. Comparison was made with gamma-irradiated animals from 60 Co source with dose rate 1,44 Gy/min. The relative biological effectiveness (RBE) was determined by comparing the regression coefficients from the linear dose translocation yield dependency. The values of the RBE coefficients were 0.8, 0.9 and 1.2, accordingly for protons, α-particles and carbon ions

  1. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    Science.gov (United States)

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  2. Visualizing the mapped ion pathway through the Na,K-ATPase pump.

    Science.gov (United States)

    Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo; Gadsby, David C

    2009-11-01

    translocation by the Na(+),K(+)-ATPase and by other P-type pumps like the Ca(2+)- and H(+),K(+)-ATPases.

  3. Visualizing the mapped ion pathway through the Na,K-ATPase pump

    Science.gov (United States)

    Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo; Gadsby, David C.

    2009-01-01

    The Na+,K+-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na+ ions for extracellular K+ ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na+,K+-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na+ current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na+,K+-ATPase based on the structure of the related sarcoplasmic- and endoplasmic-reticulum (SERCA) Ca2+-ATPase in a BeF3−-trapped state1,2, in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4, and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 which forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by

  4. Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling.

    Science.gov (United States)

    Seeger, Markus A; von Ballmoos, Christoph; Verrey, François; Pos, Klaas M

    2009-06-30

    The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily.

  5. Effect of endurance swimming on rat cardiac myofibrillar ATPase with experimental diabetes.

    Science.gov (United States)

    Belcastro, A N; Maybank, P; Rossiter, M; Secord, D

    1985-09-01

    Diabetes is characterized by depressed cardiac functional properties attributed to Ca2+-activated ATPase activity. In contrast, endurance swimming enhances the cardiac functional properties and Ca2+-activated myofibril ATPase. Thus, the purpose of this study was to observe if the changes associated with experimental diabetes can be ameliorated with training. Diabetes was induced with a single i.v. injection of streptozotocin (60 mg/kg). Blood and urine glucose concentrations were 802 +/- 44 and 6965 +/- 617 mg/dL, respectively. The training control and training diabetic animals were made to swim (+/- 2% body weight) 4 days/week for 8 weeks. Cardiac myofibril, at 10 microM free Ca2+ concentration was reduced by 54% in the sedentary diabetics compared with sedentary control animals (p less than 0.05). Swim training enhanced the Ca2+-activated myofibril ATPase activities for the normal animals. The diabetic animals, which swam for 8 weeks, had further reduced their Ca2+-activated myofibril ATPase activity when compared with sedentary diabetics (p less than 0.05). Similarly, the Mg2+-stimulated myofibril ATPase activity was depressed by 31% in diabetics following endurance swimming. It is concluded that the depressed Ca2+-activated myofibril ATPase activity of diabetic hearts is not reversible with endurance swimming.

  6. Proton nuclear magnetic resonance imaging of regionally ischemic canine hearts: effects of paramagnetic proton signal enhancement

    International Nuclear Information System (INIS)

    Brady, T.J.; Goldman, M.R.; Pykett, I.L.; Buonanno, F.S.; Kistler, J.P.; Newhouse, J.H.; Burt, C.T.; Hinshaw, W.S.; Pohost, G.M.

    1982-01-01

    In a study to evaluate the potential of proton nuclear magnetic resonance (NMR) imaging with and without manganese contrast enhancement for detecting acute myocardial infarction, 12 dogs underwent 90-minute occlusion of the left circumflex coronary artery. Transverse-section NMR images of the excised, nonbeating heart were obtained at 1-cm intervals using the steady-state-free-precession (SSFP) technique. All NMR images revealed detailed structure of the heart. The three hearts without manganese showed no difference in intensity between the normal and the ischemic posterior regions, whereas those with manganese demonstrated a clearly demarcated zone of reduced signal intensity consistent with the ischemic zone. It is concluded that high-resolution tomograms of the excised canine myocardium can be obtained using proton NMR imaging. With the SSFP imaging technique, proton signal enhancement with manganese infusion is necessary to differentiate between ischemic and nonischemic myocardium after 90 minutes of coronary occlusion

  7. From nanochannel-induced proton conduction enhancement to a nanochannel-based fuel cell.

    Science.gov (United States)

    Liu, Shaorong; Pu, Qiaosheng; Gao, Lin; Korzeniewski, Carol; Matzke, Carolyn

    2005-07-01

    The apparent proton conductivity inside a nanochannel can be enhanced by orders of magnitude due to the electric double layer overlap. A nanochannel filled with an acidic solution is thus a micro super proton conductor, and an array of such nanochannels forms an excellent proton conductive membrane. Taking advantage of this effect, a new class of proton exchange membrane is developed for micro fuel cell applications.

  8. Na+-K+-ATPase in rat skeletal muscle: muscle fiber-specific differences in exercise-induced changes in ion affinity and maximal activity

    DEFF Research Database (Denmark)

    Juel, Carsten

    2008-01-01

    It is unclear whether muscle activity reduces or increases Na(+)-K(+)-ATPase maximal in vitro activity in rat skeletal muscle, and it is not known whether muscle activity changes the Na(+)-K(+)-ATPase ion affinity. The present study uses quantification of ATP hydrolysis to characterize muscle fiber...... membranes of glycolytic muscle, which abolished the fiber-type difference in Na(+) affinity. K(m) for K(+) (in the presence of Na(+)) was not influenced by running. Running only increased the maximal in vitro activity (V(max)) in total membranes from soleus, whereas V(max) remained constant in the three...... other muscles tested. In conclusion, muscle activity induces fiber type-specific changes both in Na(+) affinity and maximal in vitro activity of the Na(+)-K(+)-ATPase. The underlying mechanisms may involve translocation of subunits and increased association between PLM units and the alphabeta complex...

  9. Protonation enhancement by dichloromethane doping in low-pressure photoionization.

    Science.gov (United States)

    Shu, Jinian; Zou, Yao; Xu, Ce; Li, Zhen; Sun, Wanqi; Yang, Bo; Zhang, Haixu; Zhang, Peng; Ma, Pengkun

    2016-12-01

    Doping has been used to enhance the ionization efficiency of analytes in atmospheric pressure photoionization, which is based on charge exchange. Compounds with excellent ionization efficiencies are usually chosen as dopants. In this paper, we report a new phenomenon observed in low-pressure photoionization: Protonation enhancement by dichloromethane (CH 2 Cl 2 ) doping. CH 2 Cl 2 is not a common dopant due to its high ionization energy (11.33 eV). The low-pressure photoionization source was built using a krypton VUV lamp that emits photons with energies of 10.0 and 10.6 eV and was operated at ~500-1000 Pa. Protonation of water, methanol, ethanol, and acetaldehyde was respectively enhanced by 481.7 ± 122.4, 197.8 ± 18.8, 87.3 ± 7.8, and 93.5 ± 35.5 times after doping 291 ppmv CH 2 Cl 2 , meanwhile CH 2 Cl 2 almost does not generate noticeable ions itself. This phenomenon has not been documented in the literature. A new protonation process involving in ion-pair and H-bond formations was proposed to expound the phenomenon. The observed phenomenon opens a new prospect for the improvement of the detection efficiency of VUV photoionization.

  10. Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex.

    Science.gov (United States)

    Bodnar, Nicholas O; Rapoport, Tom A

    2017-05-04

    The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tübingen (Germany)

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  12. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Peter Rohloff

    Full Text Available Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+/H(+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

  13. Glutamate Water Gates in the Ion Binding Pocket of Na(+) Bound Na(+), K(+)-ATPase

    DEFF Research Database (Denmark)

    Han, Minwoo; Kopec, Wojciech; Solov'yov, Ilia A

    2017-01-01

    III is always protonated. Glutamic acid residues in the three binding sites act as water gates, and their deprotonation triggers water entry to the binding sites. From DFT calculations of Na(+) binding energies, we conclude that three protons in the binding site are needed to effectively bind Na......The dynamically changing protonation states of the six acidic amino acid residues in the ion binding pocket of the Na(+), K(+) -ATPase (NKA) during the ion transport cycle are proposed to drive ion binding, release and possibly determine Na(+) or K(+) selectivity. We use molecular dynamics (MD......(+) from water and four are needed to release them in the next step. Protonation of Asp926 in site III will induce Na(+) release, and Glu327, Glu954 and Glu779 are all likely to be protonated in the Na(+) bound occluded conformation. Our data provides key insights into the role of protons in the Na...

  14. Single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Ulaszewski, S.; Van Herck, J.C.; Dufour, J.P.; Kulpa, J.; Nieuwenhuis, B.; Goffeau, A.

    1987-01-01

    A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma- 32 P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors

  15. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Science.gov (United States)

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K

    2014-01-01

    Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  16. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Directory of Open Access Journals (Sweden)

    Nidhi Thakur

    Full Text Available BACKGROUND: Expression of double strand RNA (dsRNA designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi, thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci upon oral feeding. The v-ATPase subunit A (v-ATPaseA coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  17. Constitutive activation of a plasma membrane H+-ATPase prevents abscisic acid-mediated stomatal closure

    Science.gov (United States)

    Merlot, Sylvain; Leonhardt, Nathalie; Fenzi, Francesca; Valon, Christiane; Costa, Miguel; Piette, Laurie; Vavasseur, Alain; Genty, Bernard; Boivin, Karine; Müller, Axel; Giraudat, Jérôme; Leung, Jeffrey

    2007-01-01

    Light activates proton (H+)-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO2 to photosynthetic tissues. Light to darkness transition, high CO2 levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H+-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO2 and darkness. The OST2 gene encodes the major plasma membrane H+-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H+-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure. PMID:17557075

  18. Membrane Targeting of P-type ATPases in Plant Cells

    International Nuclear Information System (INIS)

    Harper, Jeffrey F.

    2004-01-01

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

  19. Single-molecule analysis of inhibitory pausing states of V1-ATPase.

    Science.gov (United States)

    Uner, Naciye Esma; Nishikawa, Yoshihiro; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

    2012-08-17

    V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V(1)-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V(1) in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F(1), suggesting that LP represents the ADP-inhibited state of V(1)-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V(1)-ATPase rotation modulates the off rate of ADP.

  20. The virtual enhancements - solar proton event radiation (VESPER) model

    Science.gov (United States)

    Aminalragia-Giamini, Sigiava; Sandberg, Ingmar; Papadimitriou, Constantinos; Daglis, Ioannis A.; Jiggens, Piers

    2018-02-01

    A new probabilistic model introducing a novel paradigm for the modelling of the solar proton environment at 1 AU is presented. The virtual enhancements - solar proton event radiation model (VESPER) uses the European space agency's solar energetic particle environment modelling (SEPEM) Reference Dataset and produces virtual time-series of proton differential fluxes. In this regard it fundamentally diverges from the approach of existing SPE models that are based on probabilistic descriptions of SPE macroscopic characteristics such as peak flux and cumulative fluence. It is shown that VESPER reproduces well the dataset characteristics it uses, and further comparisons with existing models are made with respect to their results. The production of time-series as the main output of the model opens a straightforward way for the calculation of solar proton radiation effects in terms of time-series and the pairing with effects caused by trapped radiation and galactic cosmic rays.

  1. Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

    Science.gov (United States)

    Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

    2003-12-05

    Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

  2. Limited uptake, translocation and enhanced metabolic degradation contribute to glyphosate tolerance in Mucuna pruriens var. utilis plants.

    Science.gov (United States)

    Rojano-Delgado, Antonia María; Cruz-Hipolito, Hugo; De Prado, Rafael; Luque de Castro, María Dolores; Franco, Antonio Rodríguez

    2012-01-01

    Velvet bean (Mucuna pruriens, Fabaceae) plants exhibits an innate, very high resistance (i.e., tolerance) to glyphosate similar to that of plants which have acquired resistance to this herbicide as a trait. We analyzed the uptake of [(14)C]-glyphosate by leaves and its translocation to meristematic tissues, and used scanning electron micrographs to further analyze the cuticle and 3D capillary electrophoresis to investigate a putative metabolism capable of degrading the herbicide. Velvet bean exhibited limited uptake of glyphosate and impaired translocation of the compound to meristematic tissues. Also, for the first time in a higher plant, two concurrent pathways capable of degrading glyphosate to AMPA, Pi, glyoxylate, sarcosine and formaldehyde as end products were identified. Based on the results, the innate tolerance of velvet bean to glyphosate is possibly a result of the combined action of the previous three traits, namely: limited uptake, impaired translocation and enhanced degradation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

    Science.gov (United States)

    Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

    2016-11-01

    Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

  4. Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.).

    Science.gov (United States)

    Mirza, Neelofar; Taj, Gohar; Arora, Sandeep; Kumar, Anil

    2014-10-25

    Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca(2+)/H(+) antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca(2+) ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca(2+) ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Enhanced proton acceleration by ultrashort laser pulse interaction with nanostructured thin films

    International Nuclear Information System (INIS)

    Mondal, Angana; Dalui, Malay; Tata, Sheroy; Sarkar, Subhrangshu; Jha, Jagannath; Lad, Amit; Krishnamurthy, M.; Ayyub, P.; Wang, W m; Sheng, Z m

    2015-01-01

    Enhancement of local electromagnetic field in nanostructured targets as opposed to plain polished targets has been experimentally observed and studied. This increase in field strength leads to enhanced hot electron generation, which gives rise to highly energetic ions through Target Normal Sheath Acceleration. As the laser energy coupled to the electrons increases, the sheath magnitude is expected to increase, leading to an enhancement in ion acceleration. We investigate energy enhancements in ions generated as a result of intense femtosecond laser interaction with nanostructured thin film targets, comprising 2 μm Ta foil coated with 100-200 nm diameter Ta clusters. The optimum nanoparticle size of 100 nm corresponding to maximum laser energy absorption has been predetermined through PIC simulations. The accelerated ions have been studied using Thompson parabola spectrometer at a laser intensity of 15 x 10 19 W/cm 2 at the TIFR high contrast 100 TW Ti:Sapphire laser facility. The proton cut-off energy is observed to increase rapidly with increasing cluster density till a saturation is reached. The enhancement in the proton cut-off energy is observed to be three-fold as compared to the proton cut-off energy for unstructured foils. (author)

  6. [Properties and localization of Mg- and Ca-ATpase activities in wheat embryo cell nuclei].

    Science.gov (United States)

    Vasil'eva, N A; Belkina, G G; Stepanenko, S Y; Atalykova, F I; Oparin, A I

    1978-05-01

    The isolated nuclei of wheat embryo possess the ATPase activity. The addition of Mg2+ and Ca2+ significantly increases the activities of nuclear ATPases, whereas Hg2+, Cu2+ and Mn2+ inhibit the activity. The activating effect of Mg2+ is enhanced by an addition of Na and K ions. The activity of wheat embryo nuclear Mg-ATPase is higher than its Ca-ATPase activity; both ATPases also differ in their pH optima. Separation of total nuclear protein according to the solubility of its individual protein components in wheat and strong salt solutions, using the detergents, as well as ammonium sulfate precipitation and dialysis do not result in separation of Mg-activated and Ca-activated ATPases, although their levels of activities and ratios change in the course of fractionation. The Mg- and Ca-ATPase activities of the wheat embryo nuclei were found in the nuclear fraction of albumin, in nonhistone proteins and nuclear membranes. In the albumin nuclear fraction and subfractions of non-histone proteins the higher level of activity is observed in Ca-ATPase, whereas in the nuclei and soluble fractions of residual proteins in Mg-ATPase.

  7. Enhanced Proton Conductivity in Y-Doped BaZrO3 via Strain Engineering.

    Science.gov (United States)

    Fluri, Aline; Marcolongo, Aris; Roddatis, Vladimir; Wokaun, Alexander; Pergolesi, Daniele; Marzari, Nicola; Lippert, Thomas

    2017-12-01

    The effects of stress-induced lattice distortions (strain) on the conductivity of Y-doped BaZrO 3 , a high-temperature proton conductor with key technological applications for sustainable electrochemical energy conversion, are studied. Highly ordered epitaxial thin films are grown in different strain states while monitoring the stress generation and evolution in situ. Enhanced proton conductivity due to lower activation energies is discovered under controlled conditions of tensile strain. In particular, a twofold increased conductivity is measured at 200 °C along a 0.7% tensile strained lattice. This is at variance with conclusions coming from force-field simulations or the static calculations of diffusion barriers. Here, extensive first-principles molecular dynamic simulations of proton diffusivity in the proton-trapping regime are therefore performed and found to agree with the experiments. The simulations highlight that compressive strain confines protons in planes parallel to the substrate, while tensile strain boosts diffusivity in the perpendicular direction, with the net result that the overall conductivity is enhanced. It is indeed the presence of the dopant and the proton-trapping effect that makes tensile strain favorable for proton conduction.

  8. Correlation of near-Earth proton enhancements >100 MeV with parameters of solar microwave bursts

    Science.gov (United States)

    Grechnev, Victor; Kiselev, Valentin; Meshalkina, Nataliya; Chertok, Ilya

    2017-09-01

    We analyze the relations between various combinations of peak fluxes and fluences of solar microwave bursts at 35 GHz recorded with the Nobeyama Radio Polarimeters during 1990–2015, and corresponding parameters of proton enhancements with E>100 MeV exceeding 0.1 pfu registered by GOES monitors in near-Earth environment. The highest correlation has been found between the microwave and proton fluences. This fact reflects a dependence of the total number of protons on the total duration of the acceleration process. In the events with strong flares, the correlation coefficients of proton fluences with microwave and soft X-ray fluences are higher than those with speeds of coronal mass ejections. The results indicate a statistically larger contribution of flare processes to acceleration of high-energy protons. Acceleration by shock waves seems to be less important at high energies in events associated with strong flares, although its contribution probably prevails in weaker events. The probability of a detectable proton enhancement was found to directly depend on the peak flux and duration of a microwave burst. This can be used for diagnostics of proton enhancements based on microwave observations.

  9. Quantitative investigation of physical factors contributing to gold nanoparticle-mediated proton dose enhancement

    International Nuclear Information System (INIS)

    Cho, Jongmin; Manohar, Nivedh; Kerr, Matthew; Cho, Sang Hyun; Gonzalez-Lepera, Carlos; Krishnan, Sunil

    2016-01-01

    Some investigators have shown tumor cell killing enhancement in vitro and tumor regression in mice associated with the loading of gold nanoparticles (GNPs) before proton treatments. Several Monte Carlo (MC) investigations have also demonstrated GNP-mediated proton dose enhancement. However, further studies need to be done to quantify the individual physical factors that contribute to the dose enhancement or cell-kill enhancement (or radiosensitization). Thus, the current study investigated the contributions of particle-induced x-ray emission (PIXE), particle-induced gamma-ray emission (PIGE), Auger and secondary electrons, and activation products towards the total dose enhancement. Specifically, GNP-mediated dose enhancement was measured using strips of radiochromic film that were inserted into vials of cylindrical GNPs, i.e. gold nanorods (GNRs), dispersed in a saline solution (0.3 mg of GNRs/g or 0.03% of GNRs by weight), as well as vials containing water only, before proton irradiation. MC simulations were also performed with the tool for particle simulation code using the film measurement setup. Additionally, a high-purity germanium detector system was used to measure the photon spectrum originating from activation products created from the interaction of protons and spherical GNPs present in a saline solution (20 mg of GNPs/g or 2% of GNPs by weight). The dose enhancement due to PIXE/PIGE recorded on the films in the GNR-loaded saline solution was less than the experimental uncertainty of the film dosimetry (<2%). MC simulations showed highly localized dose enhancement (up to a factor 17) in the immediate vicinity (<100 nm) of GNRs, compared with hypothetical water nanorods (WNRs), mostly due to GNR-originated Auger/secondary electrons; however, the average dose enhancement over the entire GNR-loaded vial was found to be minimal (0.1%). The dose enhancement due to the activation products from GNPs was minimal (<0.1%) as well. In conclusion, under the

  10. Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome.

    Science.gov (United States)

    Cho, Y. W.

    1995-01-01

    To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium. PMID:7598829

  11. LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

    Science.gov (United States)

    Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

    2016-01-13

    Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

  12. Iron overload impact on P-ATPases.

    Science.gov (United States)

    Sousa, Leilismara; Pessoa, Marco Tulio C; Costa, Tamara G F; Cortes, Vanessa F; Santos, Herica L; Barbosa, Leandro Augusto

    2018-03-01

    Iron is a chemical element that is active in the fundamental physiological processes for human life, but its burden can be toxic to the body, mainly because of the stimulation of membrane lipid peroxidation. For this reason, the action of iron on many ATPases has been studied, especially on P-ATPases, such as the Na + ,K + -ATPase and the Ca 2+ -ATPase. On the Fe 2+ -ATPase activity, the free iron acts as an activator, decreasing the intracellular Fe 2+ and playing a protection role for the cell. On the Ca 2+ -ATPase activity, the iron overload decreases the enzyme activity, raising the cytoplasmic Ca 2+ and decreasing the sarco/endoplasmic reticulum and the Golgi apparatus Ca 2+ concentrations, which could promote an enzyme oxidation, nitration, and fragmentation. However, the iron overload effect on the Na + ,K + -ATPase may change according to the tissue expressions. On the renal cells, as well as on the brain and the heart, iron promotes an enzyme inactivation, whereas its effect on the erythrocytes seems to be the opposite, directly stimulating the ATPase activity, or stimulating it by signaling pathways involving ROS and PKC. Modulations in the ATPase activity may impair the ionic transportation, which is essential for cell viability maintenance, inducing irreversible damage to the cell homeostasis. Here, we will discuss about the iron overload effect on the P-ATPases, such as the Na + ,K + -ATPase, the Ca 2+ -ATPase, and the Fe 2+ -ATPase.

  13. Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

    Science.gov (United States)

    Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

    2015-04-01

    The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Evolution of plant P-type ATPases

    Directory of Open Access Journals (Sweden)

    Christian N.S. Pedersen

    2012-02-01

    Full Text Available Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauria and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a moss, Selaginella moellendorffii (a primitive vascular plant, and Arabidopsis thaliana (a model flowering plant. Each organism contained sequences for all five subfamilies of P-type ATPases. Our analysis demonstrates when specific subgroups of P-type ATPases disappeared in the evolution of Angiosperms. Na/K-pump related P2C ATPases were lost with the evolution of streptophytes whereas Na+ or K+ pumping P2D ATPases and secretory pathway Ca2+-ATPases remained until mosses. An N-terminally located calmodulin binding domain in P2B ATPases can only be detected in pumps from Streptophytae, whereas, like in animals, a C-terminally localized calmodulin binding domain might be present in chlorophyte P2B Ca2+-ATPases. Chlorophyte genomes encode P3A ATPases resembling protist plasma membrane H+-ATPases and a C-terminal regulatory domain is missing. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps.

  15. The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism.

    Science.gov (United States)

    Dance, Ian

    2015-11-07

    Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O(+) from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 Å) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-HO hydrogen bonds, and the occurrence of long (up to 3.6 Å) separations between contiguous water molecules. Both steps involve low potential energy barriers, chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The largest potential barrier is estimated to be 14 kcal mol(-1). These results are discussed in the context of reactivity data for nitrogenase

  16. The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability.

    Directory of Open Access Journals (Sweden)

    Tobias Wilms

    2017-06-01

    Full Text Available The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

  17. Spatial distributions of dose enhancement around a gold nanoparticle at several depths of proton Bragg peak

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Jihun [Department of Radiation Oncology, Hokkaido University Graduate School of Medicine, Hokkaido University (Japan); Sutherland, Kenneth [Department of Medical Physics, Hokkaido University Graduate School of Medicine, Hokkaido University (Japan); Hashimoto, Takayuki [Department of Radiation Medicine, Hokkaido University Graduate School of Medicine (Japan); Shirato, Hiroki [Department of Radiation Medicine, Hokkaido University Graduate School of Medicine and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University (Japan); Date, Hiroyuki, E-mail: date@hs.hokudai.ac.jp [Faculty of Health Sciences, Hokkaido University (Japan)

    2016-10-01

    Gold nanoparticles (GNPs) have been recognized as a promising candidate for a radiation sensitizer. A proton beam incident on a GNP can produce secondary electrons, resulting in an enhancement of the dose around the GNP. However, little is known about the spatial distribution of dose enhancement around the GNP, especially in the direction along the incident proton. The purpose of this study is to determine the spatial distribution of dose enhancement by taking the incident direction into account. Two steps of calculation were conducted using the Geant4 Monte Carlo simulation toolkit. First, the energy spectra of 100 and 195 MeV protons colliding with a GNP were calculated at the Bragg peak and three other depths around the peak in liquid water. Second, the GNP was bombarded by protons with the obtained energy spectra. Radial dose distributions were computed along the incident beam direction. The spatial distributions of the dose enhancement factor (DEF) and subtracted dose (D{sub sub}) were then evaluated. The spatial DEF distributions showed hot spots in the distal radial region from the proton beam axis. The spatial D{sub sub} distribution isotropically spread out around the GNP. Low energy protons caused higher and wider dose enhancement. The macroscopic dose enhancement in clinical applications was also evaluated. The results suggest that the consideration of the spatial distribution of GNPs in treatment planning will maximize the potential of GNPs.

  18. Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

    Science.gov (United States)

    Haruta, Miyoshi; Tan, Li Xuan; Bushey, Daniel B; Swanson, Sarah J; Sussman, Michael R

    2018-01-01

    A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase. © 2018 American Society of Plant Biologists. All Rights Reserved.

  19. Light induces changes in activities of Na+/K+(NH4+-ATPase, H+/K+(NH4+-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam Tridacna squamosa

    Directory of Open Access Journals (Sweden)

    Alex Y K Ip

    2015-03-01

    Full Text Available The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control, on enzymatic activities of transporters involved in the transport of NH4+ or H+, and activities of enzymes involved in converting NH4+ to glutamate/glutamine in inner mantle, outer mantle and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH4+ in substitution for K+ to activate Na+/K+-ATPase (NKA, manifested as a significant increase in the Na+/NH4+-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H+ released from CaCO3 deposition could react with NH3 to form NH4+ in the extrapallial fluid, and NH4+ could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH4+ and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM-sensitive-V-H+-ATPase (VATPase in the inner mantle, and significant increases in the Na+/K+-activated-NKA, H+/NH4+-activated-H+/K+-ATPase and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na+ homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH4+ from the environment. This is the first report on light having direct enhancing effects on activities of certain

  20. Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase.

    Science.gov (United States)

    Cornelius, Flemming; Tsunekawa, Naoki; Toyoshima, Chikashi

    2018-02-09

    The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis-powered Na + and K + electrochemical gradients across the cell membrane. These ion gradients drive co- and countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na + and K + , despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2·Pi·2K + and Na + -bound E1∼P·ADP suggest that the dimensions of the respective binding sites in Na,K-ATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K + at the extracellular face ( i.e. E2 conformation) as the pH is lowered from 7.5 to 5. We also observed a smaller increase in affinity for K + on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na + Theoretical analysis of the p K a values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na + /K + selectivities may arise from the protonation or ionization of key residues. The increase in K + selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp 808 protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    Science.gov (United States)

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner.

    Science.gov (United States)

    Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

    2015-01-01

    Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H(+)-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

  3. The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase.

    Science.gov (United States)

    Bredeston, Luis M; González Flecha, F Luis

    2016-07-01

    Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG(‡)=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH(‡)=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH(‡)=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. NCBI nr-aa BLAST: CBRC-DYAK-03-0005 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-03-0005 ref|ZP_01001263.1| copper-translocating P-type ATPase [Oceanicola bats...01368.1| copper-translocating P-type ATPase [Oceanicola batsensis HTCC2597] gb|EAU47853.1| copper-translocating P-type ATPase [Roseovarius sp. HTCC2601] ZP_01001263.1 0.16 26% ...

  5. Na(+),K (+)-ATPase as a docking station: protein-protein complexes of the Na(+),K (+)-ATPase.

    Science.gov (United States)

    Reinhard, Linda; Tidow, Henning; Clausen, Michael J; Nissen, Poul

    2013-01-01

    The Na(+),K(+)-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na(+) ions out of the cell and of K(+) ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na(+),K(+)-ATPase, recent work has suggested additional roles for Na(+),K(+)-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na(+),K(+)-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na(+),K(+)-ATPase as a signal transducer, but also briefly discuss other Na(+),K(+)-ATPase protein-protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.

  6. A pivotal role of vacuolar H(+)-ATPase in regulation of lipid production in Phaeodactylum tricornutum.

    Science.gov (United States)

    Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

    2016-08-08

    Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H(+)-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1.

  7. Reactivity of 12-tungstophosphoric acid and its inhibitor potency toward Na+/K+-ATPase: A combined 31P NMR study, ab initio calculations and crystallographic analysis.

    Science.gov (United States)

    Bošnjaković-Pavlović, Nada; Bajuk-Bogdanović, Danica; Zakrzewska, Joanna; Yan, Zeyin; Holclajtner-Antunović, Ivanka; Gillet, Jean-Michel; Spasojević-de Biré, Anne

    2017-11-01

    Influence of 12-tungstophosphoric acid (WPA) on conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) in the presence of Na + /K + -ATPase was monitored by 31 P NMR spectroscopy. It was shown that WPA exhibits inhibitory effect on Na + /K + -ATPase activity. In order to study WPA reactivity and intermolecular interactions between WPA oxygen atoms and different proton donor types (D=O, N, C), we have considered data for WPA based compounds from the Cambridge Structural Database (CSD), the Crystallographic Open Database (COD) and the Inorganic Crystal Structure Database (ICSD). Binding properties of Keggin's anion in biological systems are illustrated using Protein Data Bank (PDB). This work constitutes the first determination of theoretical Bader charges on polyoxotungstate compound via the Atom In Molecule theory. An analysis of electrostatic potential maps at the molecular surface and charge of WPA, resulting from DFT calculations, suggests that the preferred protonation site corresponds to WPA bridging oxygen. These results enlightened WPA chemical reactivity and its potential biological applications such as the inhibition of the ATPase activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

    International Nuclear Information System (INIS)

    Miles, Andrew J.; Fedosova, Natalya U.; Hoffmann, Søren V.; Wallace, B.A.; Esmann, Mikael

    2013-01-01

    Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography

  9. Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Andrew J. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Fedosova, Natalya U. [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark); Hoffmann, Søren V. [ISA, Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus (Denmark); Wallace, B.A. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Esmann, Mikael, E-mail: me@biophys.au.dk [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark)

    2013-05-31

    Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

  10. Effect of ionizing radiation on Ca2+-ATPase and Mg2+-ATPase: the role of ligands

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1994-01-01

    The change of Ca 2+ -ATPase and Mg 2+ -ATPase activity in plasma membranes of thymocytes irradiated with doses of 10 2 , 10 3 and 10 4 Gy in the presence of Ca 2+ , Mg 2+ and ATP was studied. Stabilizing effect of Ca 2+ and Mg 2+ on Ca 2+ -ATPase and ATP on Mg 2+ -ATPase under irradiation was established

  11. Problem-elephant translocation: translocating the problem and the elephant?

    Directory of Open Access Journals (Sweden)

    Prithiviraj Fernando

    Full Text Available Human-elephant conflict (HEC threatens the survival of endangered Asian elephants (Elephas maximus. Translocating "problem-elephants" is an important HEC mitigation and elephant conservation strategy across elephant range, with hundreds translocated annually. In the first comprehensive assessment of elephant translocation, we monitored 16 translocations in Sri Lanka with GPS collars. All translocated elephants were released into national parks. Two were killed within the parks where they were released, while all the others left those parks. Translocated elephants showed variable responses: "homers" returned to the capture site, "wanderers" ranged widely, and "settlers" established home ranges in new areas soon after release. Translocation caused wider propagation and intensification of HEC, and increased elephant mortality. We conclude that translocation defeats both HEC mitigation and elephant conservation goals.

  12. RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus

    Directory of Open Access Journals (Sweden)

    Jill M. Perreira

    2015-08-01

    Full Text Available Human rhinovirus (HRV causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.

  13. Mechanism of photoinactivation of plant plasma membrane ATPases

    International Nuclear Information System (INIS)

    Imbrie, C.W.; Murphy, T.M.

    1984-01-01

    UV radiation at 290 and 365 nm inactivates two forms of the K + -stimulated ATPase associated with the plasma membrane of suspension-cultured cells of Rosa damascena. One form is 15 and 36 times more sensitive than the other to 290 and 365 nm, respectively. For both forms, the inactivation requires oxygen, is inhibited by azide and diazobicyclo(2.2.2.2)octane, but not glycerol, and is enhanced up to 7.5 times in deuterium oxide solvent. Inactivation occurs concomitantly with loss of absorbance at 290 nm. Cs + and NO 3 - , quenchers of tryptophan fluorescence, inhibit inactivation. The results suggest that inactivation involves singlet-oxygen mediated destruction of tryptophans in the ATPases. (author)

  14. Myocardial Na,K-ATPase: Clinical aspects

    OpenAIRE

    Kjeldsen, Keld

    2003-01-01

    The specific binding of digitalis glycosides to Na,K-ATPase is used as a tool for Na,K-ATPase quantification with high accuracy and precision. In myocardial biopsies from patients with heart failure, total Na,K-ATPase concentration is decreased by around 40%; a correlation exists between a decrease in heart function and a decrease in Na,K-ATPase concentration. During digitalization, around 30% of remaining pumps are occupied by digoxin. Myocardial Na,K-ATPase is also influenced by other drugs...

  15. Active transport of Na+ by reconstituted Na,K-ATPase

    International Nuclear Information System (INIS)

    Boldyrev, A.A.; Svinukhova, I.A.

    1987-01-01

    The ability of ATP, CTP, ITP, GTP, and UTP to support ouabain-sensitive accumulation of Na + by proteoliposomes with a reconstituted Na/K-pump was investigated. At a low [Na + ]/[K + ] ratio in the medium (20 mM/50 mM), a correlation is observed between the proton-accepting capacity of the nucleotide and its effectiveness as a substrate of active transport. To test the hypothesis of the importance of the presence of a negative charge in the 1-position of the purine (3-pyrimidine) base of the nucleotide for mutual transitions between the Na- and K-conformations of Na,K-ATPase they used two analogs of ATP: N 1 -hydroxy-ATP, possessing proton acceptor capacity, and N 1 -methoxy-ATP, in the molecule of which the negative charge is quenched by a methyl group. The first substrate supports active accumulation of Na + in proteoliposomes at the same rate as ATP, whereas the second substrate is relatively ineffective

  16. Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia.

    Science.gov (United States)

    Lestari, Silvia W; Miati, Dessy Noor; Seoharso, P; Sugiyanto, R; Pujianto, Dwi A

    2017-10-01

    Asthenozoospermia, which is characterized by reduced motility, is one of the etiologies of male infertility. Its biochemical and functional consequences include altered ATPase activity. This study investigated the activities of Na + , K + -ATPase and Ca 2+ -ATPase and the expression of Na + , K + -ATPase α4 and PMCA4 isoforms in human sperm of asthenozoospermic infertile men. Nineteen samples from asthenozoospermic infertile couples were examined in this study. Computerized-assisted semen analysis (CASA) was performed, and the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The Na + , K + -ATPase α4 and PMCA4 isoform expression levels were measured by western immunoblotting, whereas the protein distribution was examined by immunocytochemistry. This showed that the Na + , K + -ATPase activity and the Na + , K + -ATPase α4 isoform expression were lower in the asthenozoospermia group than in the normozoospermia group (8.688±1.161 versus 13.851±1.884 µmol Pi/mg protein/h, respectively; p>0.05). In contrast, the Ca 2+ -ATPase activity was significantly higher in the asthenozoospermia group than in the normozoospermia group (11.154±1.186 versus 2.725±0.545 µmol Pi/mg protein/h, respectively; p0.05). The altered ATPase activity and isoform expression in asthenozoospermia may impair sperm structure and function.

  17. Rescue of Na+ and H+ binding in Na+,K+-ATPase M8 aspartate mutants by secondary mutation

    DEFF Research Database (Denmark)

    Holm, Rikke; Einholm, Anja P.; Andersen, Jens Peter

    A mutation replacing the aspartate in transmembrane segment M8 in the a3-isoform of Na,K-ATPase with asparagine has been found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood. This aspartate may be a critical Na+ coordinating residue, but the crystal......-isoforms of Na,K-ATPase, and much smaller effects were seen for other mutations to the M8 aspartate, which were less disruptive of Na+ binding than mutations to other residues related to Na+ site III. The D928 (rat a1 numbering) mutations strongly diminished the cooperativity of Na+ binding. Moreover the p......H optimum of Na,K-ATPase activity was left-shifted, again with D928N being most disruptive. The reduced affinity for activating Na+ and for inhibitory protons, caused by D928N and D928A mutations, could be rescued by introduction of an additional mutation of a glutamate located far away from D928....

  18. Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase.

    Science.gov (United States)

    Stanley, Kevin S; Meyer, Dylan J; Gatto, Craig; Artigas, Pablo

    2016-12-06

    The Na + ,K + -ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na + i ) for two extracellular K+ (K + o ) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na + ] o and [K + ] o , the phosphorylated E2P conformation passively imports protons generating an inward current (I H ), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of I H , we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on I H and, for comparison, on transient currents measured at normal Na + o (Q Na ). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na + i , I H and Q Na were both activated by ATP, but not ADP; 2) the [Na + ] i dependence of I H in saturating ATP showed K 0.5,Na  = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na + ] i yielded K 0.5,ATP  = 48 ± 11 μM (in comparison, Na + i -dependent Q Na yields K 0.5,Na  = 0.8 ± 0.2 mM and K 0.5,ATP  = 0.43 ± 0.03 μM; 3) ATP activated I H in the presence of K + i (∼15% of the I H observed in Na + i ) only when Mg 2+ i was also present; and 4) beryllium fluoride induced maximal I H  even in the absence of nucleotide. These data indicate that I H occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na + i and ATP, or by backward binding of K + i to E1, which drives the pump to the occluded E2(2K), where free P i (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K + by backdoor NKA phosphorylation. Maximal I H through beryllium-fluorinated NKA indicates that this complex mimics ATP

  19. Effects of Na/K-ATPase and its ligands on bone marrow stromal cell differentiation

    Directory of Open Access Journals (Sweden)

    Moustafa Sayed

    2014-07-01

    Full Text Available Endogenous ligands of Na/K-ATPase have been demonstrated to increase in kidney dysfunction and heart failure. It is also reported that Na/K-ATPase signaling function effects stem cell differentiation. This study evaluated whether Na/K-ATPase activation through its ligands and associated signaling functions affect bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells differentiation capacity. BMSCs were isolated from male Sprague–Dawley rats and cultured in minimal essential medium alpha (MEM-α supplemented with 15% Fetal Bovine serum (FBS. The results showed that marinobufagenin (MBG, a specific Na/K-ATPase ligand, potentiated rosiglitazone-induced adipogenesis in these BMSCs. Meanwhile, it attenuated BMSC osteogenesis. Mechanistically, MBG increased CCAAT/enhancer binding protein alpha (C/EBPα protein expression through activation of an extracellular regulated kinase (ERK signaling pathway, which leads to enhanced rosiglitazone-induced adipogenesis. Inhibition of ERK activation by U0126 blocks the effect of MBG on C/EBPα expression and on rosiglitazone-induced adipogenesis. Reciprocally, MBG reduced runt-related transcription factor 2 (RunX2 expression, which resulted in the inhibition of osteogenesis induced by β-glycerophosphate/ascorbic acid. MBG also potentiated rosiglitazone-induced adipogenesis in 3T3-L1 cells and in mouse BMSCs. These results suggest that Na/K-ATPase and its signaling functions are involved in the regulation of BMSCs differentiation.

  20. Enhanced production of multi-strange hadrons in high-multiplicity proton–proton collisions

    CERN Document Server

    Adam, Jaroslav; Aggarwal, Madan Mohan; Aglieri Rinella, Gianluca; Agnello, Michelangelo; Agrawal, Neelima; Ahammed, Zubayer; Ahmad, Shakeel; Ahn, Sang Un; Aiola, Salvatore; Akindinov, Alexander; Alam, Sk Noor; Silva De Albuquerque, Danilo; Aleksandrov, Dmitry; Alessandro, Bruno; Alexandre, Didier; Alfaro Molina, Jose Ruben; Alici, Andrea; Alkin, Anton; Alme, Johan; Alt, Torsten; Altinpinar, Sedat; Altsybeev, Igor; Alves Garcia Prado, Caio; An, Mangmang; Andrei, Cristian; Andrews, Harry Arthur; Andronic, Anton; Anguelov, Venelin; Anticic, Tome; Antinori, Federico; Antonioli, Pietro; Aphecetche, Laurent Bernard; Appelshaeuser, Harald; Arcelli, Silvia; Arnaldi, Roberta; Arnold, Oliver Werner; Arsene, Ionut Cristian; Arslandok, Mesut; Audurier, Benjamin; Augustinus, Andre; Averbeck, Ralf Peter; Azmi, Mohd Danish; Badala, Angela; Baek, Yong Wook; Bagnasco, Stefano; Bailhache, Raphaelle Marie; Bala, Renu; Balasubramanian, Supraja; Baldisseri, Alberto; Baral, Rama Chandra; Barbano, Anastasia Maria; Barbera, Roberto; Barile, Francesco; Barnafoldi, Gergely Gabor; Barnby, Lee Stuart; Ramillien Barret, Valerie; Bartalini, Paolo; Barth, Klaus; Bartke, Jerzy Gustaw; Bartsch, Esther; Basile, Maurizio; Bastid, Nicole; Basu, Sumit; Bathen, Bastian; Batigne, Guillaume; Batista Camejo, Arianna; Batyunya, Boris; Batzing, Paul Christoph; Bearden, Ian Gardner; Beck, Hans; Bedda, Cristina; Behera, Nirbhay Kumar; Belikov, Iouri; Bellini, Francesca; Bello Martinez, Hector; Bellwied, Rene; Belmont Iii, Ronald John; Belmont Moreno, Ernesto; Espinoza Beltran, Lucina Gabriela; Belyaev, Vladimir; Bencedi, Gyula; Beole, Stefania; Berceanu, Ionela; Bercuci, Alexandru; Berdnikov, Yaroslav; Berenyi, Daniel; Bertens, Redmer Alexander; Berzano, Dario; Betev, Latchezar; Bhasin, Anju; Bhat, Inayat Rasool; Bhati, Ashok Kumar; Bhattacharjee, Buddhadeb; Bhom, Jihyun; Bianchi, Livio; Bianchi, Nicola; Bianchin, Chiara; Bielcik, Jaroslav; Bielcikova, Jana; Bilandzic, Ante; Biro, Gabor; Biswas, Rathijit; Biswas, Saikat; Bjelogrlic, Sandro; Blair, Justin Thomas; Blau, Dmitry; Blume, Christoph; Bock, Friederike; Bogdanov, Alexey; Boggild, Hans; Boldizsar, Laszlo; Bombara, Marek; Bonora, Matthias; Book, Julian Heinz; Borel, Herve; Borissov, Alexander; Borri, Marcello; Bossu, Francesco; Botta, Elena; Bourjau, Christian; Braun-Munzinger, Peter; Bregant, Marco; Breitner, Timo Gunther; Broker, Theo Alexander; Browning, Tyler Allen; Broz, Michal; Brucken, Erik Jens; Bruna, Elena; Bruno, Giuseppe Eugenio; Budnikov, Dmitry; Buesching, Henner; Bufalino, Stefania; Buncic, Predrag; Busch, Oliver; Buthelezi, Edith Zinhle; Bashir Butt, Jamila; Buxton, Jesse Thomas; Cabala, Jan; Caffarri, Davide; Cai, Xu; Caines, Helen Louise; Calero Diaz, Liliet; Caliva, Alberto; Calvo Villar, Ernesto; Camerini, Paolo; Carena, Francesco; Carena, Wisla; Carnesecchi, Francesca; Castillo Castellanos, Javier Ernesto; Castro, Andrew John; Casula, Ester Anna Rita; Ceballos Sanchez, Cesar; Cepila, Jan; Cerello, Piergiorgio; Cerkala, Jakub; Chang, Beomsu; Chapeland, Sylvain; Chartier, Marielle; Charvet, Jean-Luc Fernand; Chattopadhyay, Subhasis; Chattopadhyay, Sukalyan; Chauvin, Alex; Chelnokov, Volodymyr; Cherney, Michael Gerard; Cheshkov, Cvetan Valeriev; Cheynis, Brigitte; Chibante Barroso, Vasco Miguel; Dobrigkeit Chinellato, David; Cho, Soyeon; Chochula, Peter; Choi, Kyungeon; Chojnacki, Marek; Choudhury, Subikash; Christakoglou, Panagiotis; Christensen, Christian Holm; Christiansen, Peter; Chujo, Tatsuya; Chung, Suh-Urk; Cicalo, Corrado; Cifarelli, Luisa; Cindolo, Federico; Cleymans, Jean Willy Andre; Colamaria, Fabio Filippo; Colella, Domenico; Collu, Alberto; Colocci, Manuel; Conesa Balbastre, Gustavo; Conesa Del Valle, Zaida; Connors, Megan Elizabeth; Contreras Nuno, Jesus Guillermo; Cormier, Thomas Michael; Corrales Morales, Yasser; Cortes Maldonado, Ismael; Cortese, Pietro; Cosentino, Mauro Rogerio; Costa, Filippo; Crkovska, Jana; Crochet, Philippe; Cruz Albino, Rigoberto; Cuautle Flores, Eleazar; Cunqueiro Mendez, Leticia; Dahms, Torsten; Dainese, Andrea; Danisch, Meike Charlotte; Danu, Andrea; Das, Debasish; Das, Indranil; Das, Supriya; Dash, Ajay Kumar; Dash, Sadhana; De, Sudipan; De Caro, Annalisa; De Cataldo, Giacinto; De Conti, Camila; De Cuveland, Jan; De Falco, Alessandro; De Gruttola, Daniele; De Marco, Nora; De Pasquale, Salvatore; Derradi De Souza, Rafael; Deisting, Alexander; Deloff, Andrzej; Denes, Ervin Sandor; Deplano, Caterina; Dhankher, Preeti; Di Bari, Domenico; Di Mauro, Antonio; Di Nezza, Pasquale; Di Ruzza, Benedetto; Diaz Corchero, Miguel Angel; Dietel, Thomas; Dillenseger, Pascal; Divia, Roberto; Djuvsland, Oeystein; Dobrin, Alexandru Florin; Domenicis Gimenez, Diogenes; Donigus, Benjamin; Dordic, Olja; Drozhzhova, Tatiana; Dubey, Anand Kumar; Dubla, Andrea; Ducroux, Laurent; Dupieux, Pascal; Ehlers Iii, Raymond James; Elia, Domenico; Endress, Eric; Engel, Heiko; Epple, Eliane; Erazmus, Barbara Ewa; Erdemir, Irem; Erhardt, Filip; Espagnon, Bruno; Estienne, Magali Danielle; Esumi, Shinichi; Eum, Jongsik; Evans, David; Evdokimov, Sergey; Eyyubova, Gyulnara; Fabbietti, Laura; Fabris, Daniela; Faivre, Julien; Fantoni, Alessandra; Fasel, Markus; Feldkamp, Linus; Feliciello, Alessandro; Feofilov, Grigorii; Ferencei, Jozef; Fernandez Tellez, Arturo; Gonzalez Ferreiro, Elena; Ferretti, Alessandro; Festanti, Andrea; Feuillard, Victor Jose Gaston; Figiel, Jan; Araujo Silva Figueredo, Marcel; Filchagin, Sergey; Finogeev, Dmitry; Fionda, Fiorella; Fiore, Enrichetta Maria; Floris, Michele; Foertsch, Siegfried Valentin; Foka, Panagiota; Fokin, Sergey; Fragiacomo, Enrico; Francescon, Andrea; Francisco, Audrey; Frankenfeld, Ulrich Michael; Fronze, Gabriele Gaetano; Fuchs, Ulrich; Furget, Christophe; Furs, Artur; Fusco Girard, Mario; Gaardhoeje, Jens Joergen; Gagliardi, Martino; Gago Medina, Alberto Martin; Gajdosova, Katarina; Gallio, Mauro; Duarte Galvan, Carlos; Gangadharan, Dhevan Raja; Ganoti, Paraskevi; Gao, Chaosong; Garabatos Cuadrado, Jose; Garcia-Solis, Edmundo Javier; Garg, Kunal; Gargiulo, Corrado; Gasik, Piotr Jan; Gauger, Erin Frances; Germain, Marie; Gheata, Mihaela; Ghosh, Premomoy; Ghosh, Sanjay Kumar; Gianotti, Paola; Giubellino, Paolo; Giubilato, Piero; Gladysz-Dziadus, Ewa; Glassel, Peter; Gomez Coral, Diego Mauricio; Gomez Ramirez, Andres; Sanchez Gonzalez, Andres; Gonzalez, Victor; Gonzalez Zamora, Pedro; Gorbunov, Sergey; Gorlich, Lidia Maria; Gotovac, Sven; Grabski, Varlen; Grachov, Oleg Anatolievich; Graczykowski, Lukasz Kamil; Graham, Katie Leanne; Grelli, Alessandro; Grigoras, Alina Gabriela; Grigoras, Costin; Grigoryev, Vladislav; Grigoryan, Ara; Grigoryan, Smbat; Grynyov, Borys; Grion, Nevio; Gronefeld, Julius Maximilian; Grosse-Oetringhaus, Jan Fiete; Grosso, Raffaele; Gruber, Lukas; Guber, Fedor; Guernane, Rachid; Guerzoni, Barbara; Gulbrandsen, Kristjan Herlache; Gunji, Taku; Gupta, Anik; Gupta, Ramni; Haake, Rudiger; Hadjidakis, Cynthia Marie; Haiduc, Maria; Hamagaki, Hideki; Hamar, Gergoe; Hamon, Julien Charles; Harris, John William; Harton, Austin Vincent; Hatzifotiadou, Despina; Hayashi, Shinichi; Heckel, Stefan Thomas; Hellbar, Ernst; Helstrup, Haavard; Herghelegiu, Andrei Ionut; Herrera Corral, Gerardo Antonio; Herrmann, Florian; Hess, Benjamin Andreas; Hetland, Kristin Fanebust; Hillemanns, Hartmut; Hippolyte, Boris; Horak, David; Hosokawa, Ritsuya; Hristov, Peter Zahariev; Hughes, Charles; Humanic, Thomas; Hussain, Nur; Hussain, Tahir; Hutter, Dirk; Hwang, Dae Sung; Ilkaev, Radiy; Inaba, Motoi; Incani, Elisa; Ippolitov, Mikhail; Irfan, Muhammad; Isakov, Vladimir; Ivanov, Marian; Ivanov, Vladimir; Izucheev, Vladimir; Jacak, Barbara; Jacazio, Nicolo; Jacobs, Peter Martin; Jadhav, Manoj Bhanudas; Jadlovska, Slavka; Jadlovsky, Jan; Jahnke, Cristiane; Jakubowska, Monika Joanna; Janik, Malgorzata Anna; Pahula Hewage, Sandun; Jena, Chitrasen; Jena, Satyajit; Jimenez Bustamante, Raul Tonatiuh; Jones, Peter Graham; Jusko, Anton; Kalinak, Peter; Kalweit, Alexander Philipp; Kang, Ju Hwan; Kaplin, Vladimir; Kar, Somnath; Karasu Uysal, Ayben; Karavichev, Oleg; Karavicheva, Tatiana; Karayan, Lilit; Karpechev, Evgeny; Kebschull, Udo Wolfgang; Keidel, Ralf; Keijdener, Darius Laurens; Keil, Markus; Khan, Mohammed Mohisin; Khan, Palash; Khan, Shuaib Ahmad; Khanzadeev, Alexei; Kharlov, Yury; Khatun, Anisa; Kileng, Bjarte; Kim, Do Won; Kim, Dong Jo; Kim, Daehyeok; Kim, Hyeonjoong; Kim, Jinsook; Kim, Jiyoung; Kim, Minwoo; Kim, Se Yong; Kim, Taesoo; Kirsch, Stefan; Kisel, Ivan; Kiselev, Sergey; Kisiel, Adam Ryszard; Kiss, Gabor; Klay, Jennifer Lynn; Klein, Carsten; Klein, Jochen; Klein-Boesing, Christian; Klewin, Sebastian; Kluge, Alexander; Knichel, Michael Linus; Knospe, Anders Garritt; Kobdaj, Chinorat; Kofarago, Monika; Kollegger, Thorsten; Kolozhvari, Anatoly; Kondratev, Valerii; Kondratyeva, Natalia; Kondratyuk, Evgeny; Konevskikh, Artem; Kopcik, Michal; Kour, Mandeep; Kouzinopoulos, Charalampos; Kovalenko, Oleksandr; Kovalenko, Vladimir; Kowalski, Marek; Koyithatta Meethaleveedu, Greeshma; Kralik, Ivan; Kravcakova, Adela; Krivda, Marian; Krizek, Filip; Kryshen, Evgeny; Krzewicki, Mikolaj; Kubera, Andrew Michael; Kucera, Vit; Kuhn, Christian Claude; Kuijer, Paulus Gerardus; Kumar, Ajay; Kumar, Jitendra; Kumar, Lokesh; Kumar, Shyam; Kurashvili, Podist; Kurepin, Alexander; Kurepin, Alexey; Kuryakin, Alexey; Kweon, Min Jung; Kwon, Youngil; La Pointe, Sarah Louise; La Rocca, Paola; Ladron De Guevara, Pedro; Lagana Fernandes, Caio; Lakomov, Igor; Langoy, Rune; Lapidus, Kirill; Lara Martinez, Camilo Ernesto; Lardeux, Antoine Xavier; Lattuca, Alessandra; Laudi, Elisa; Lea, Ramona; Leardini, Lucia; Lee, Seongjoo; Lehas, Fatiha; Lehner, Sebastian; Lemmon, Roy Crawford; Lenti, Vito; Leogrande, Emilia; Leon Monzon, Ildefonso; Leon Vargas, Hermes; Leoncino, Marco; Levai, Peter; Li, Shuang; Li, Xiaomei; Lien, Jorgen Andre; Lietava, Roman; Lindal, Svein; Lindenstruth, Volker; Lippmann, Christian; Lisa, Michael Annan; Ljunggren, Hans Martin; Lodato, Davide Francesco; Lonne, Per-Ivar; Loginov, Vitaly; Loizides, Constantinos; Lopez, Xavier Bernard; Lopez Torres, Ernesto; Lowe, Andrew John; Luettig, Philipp Johannes; Lunardon, Marcello; Luparello, Grazia; Lupi, Matteo; Lutz, Tyler Harrison; Maevskaya, Alla; Mager, Magnus; Mahajan, Sanjay; Mahmood, Sohail Musa; Maire, Antonin; Majka, Richard Daniel; Malaev, Mikhail; Maldonado Cervantes, Ivonne Alicia; Malinina, Liudmila; Mal'Kevich, Dmitry; Malzacher, Peter; Mamonov, Alexander; Manko, Vladislav; Manso, Franck; Manzari, Vito; Mao, Yaxian; Marchisone, Massimiliano; Mares, Jiri; Margagliotti, Giacomo Vito; Margotti, Anselmo; Margutti, Jacopo; Marin, Ana Maria; Markert, Christina; Marquard, Marco; Martin, Nicole Alice; Martinengo, Paolo; Martinez Hernandez, Mario Ivan; Martinez-Garcia, Gines; Martinez Pedreira, Miguel; Mas, Alexis Jean-Michel; Masciocchi, Silvia; Masera, Massimo; Masoni, Alberto; Mastroserio, Annalisa; Matyja, Adam Tomasz; Mayer, Christoph; Mazer, Joel Anthony; Mazzilli, Marianna; Mazzoni, Alessandra Maria; Mcdonald, Daniel; Meddi, Franco; Melikyan, Yuri; Menchaca-Rocha, Arturo Alejandro; Meninno, Elisa; Mercado-Perez, Jorge; Meres, Michal; Mhlanga, Sibaliso; Miake, Yasuo; Mieskolainen, Matti Mikael; Mikhaylov, Konstantin; Milano, Leonardo; Milosevic, Jovan; Mischke, Andre; Mishra, Aditya Nath; Mishra, Tribeni; Miskowiec, Dariusz Czeslaw; Mitra, Jubin; Mitu, Ciprian Mihai; Mohammadi, Naghmeh; Mohanty, Bedangadas; Molnar, Levente; Montano Zetina, Luis Manuel; Montes Prado, Esther; Moreira De Godoy, Denise Aparecida; Perez Moreno, Luis Alberto; Moretto, Sandra; Morreale, Astrid; Morsch, Andreas; Muccifora, Valeria; Mudnic, Eugen; Muhlheim, Daniel Michael; Muhuri, Sanjib; Mukherjee, Maitreyee; Mulligan, James Declan; Gameiro Munhoz, Marcelo; Munning, Konstantin; Munzer, Robert Helmut; Murakami, Hikari; Murray, Sean; Musa, Luciano; Musinsky, Jan; Naik, Bharati; Nair, Rahul; Nandi, Basanta Kumar; Nania, Rosario; Nappi, Eugenio; Naru, Muhammad Umair; Ferreira Natal Da Luz, Pedro Hugo; Nattrass, Christine; Rosado Navarro, Sebastian; Nayak, Kishora; Nayak, Ranjit; Nayak, Tapan Kumar; Nazarenko, Sergey; Nedosekin, Alexander; Negrao De Oliveira, Renato Aparecido; Nellen, Lukas; Ng, Fabian; Nicassio, Maria; Niculescu, Mihai; Niedziela, Jeremi; Nielsen, Borge Svane; Nikolaev, Sergey; Nikulin, Sergey; Nikulin, Vladimir; Noferini, Francesco; Nomokonov, Petr; Nooren, Gerardus; Cabanillas Noris, Juan Carlos; Norman, Jaime; Nyanin, Alexander; Nystrand, Joakim Ingemar; Oeschler, Helmut Oskar; Oh, Saehanseul; Oh, Sun Kun; Ohlson, Alice Elisabeth; Okatan, Ali; Okubo, Tsubasa; Oleniacz, Janusz; Oliveira Da Silva, Antonio Carlos; Oliver, Michael Henry; Onderwaater, Jacobus; Oppedisano, Chiara; Orava, Risto; Oravec, Matej; Ortiz Velasquez, Antonio; Oskarsson, Anders Nils Erik; Otwinowski, Jacek Tomasz; Oyama, Ken; Ozdemir, Mahmut; Pachmayer, Yvonne Chiara; Pagano, Davide; Pagano, Paola; Paic, Guy; Pal, Susanta Kumar; Palni, Prabhakar; Pan, Jinjin; Pandey, Ashutosh Kumar; Papikyan, Vardanush; Pappalardo, Giuseppe; Pareek, Pooja; Park, Woojin; Parmar, Sonia; Passfeld, Annika; Paticchio, Vincenzo; Patra, Rajendra Nath; Paul, Biswarup; Pei, Hua; Peitzmann, Thomas; Peng, Xinye; Pereira Da Costa, Hugo Denis Antonio; Peresunko, Dmitry Yurevich; Perez Lezama, Edgar; Peskov, Vladimir; Pestov, Yury; Petracek, Vojtech; Petrov, Viacheslav; Petrovici, Mihai; Petta, Catia; Piano, Stefano; Pikna, Miroslav; Pillot, Philippe; Ozelin De Lima Pimentel, Lais; Pinazza, Ombretta; Pinsky, Lawrence; Piyarathna, Danthasinghe; Ploskon, Mateusz Andrzej; Planinic, Mirko; Pluta, Jan Marian; Pochybova, Sona; Podesta Lerma, Pedro Luis Manuel; Poghosyan, Martin; Polishchuk, Boris; Poljak, Nikola; Poonsawat, Wanchaloem; Pop, Amalia; Poppenborg, Hendrik; Porteboeuf, Sarah Julie; Porter, R Jefferson; Pospisil, Jan; Prasad, Sidharth Kumar; Preghenella, Roberto; Prino, Francesco; Pruneau, Claude Andre; Pshenichnov, Igor; Puccio, Maximiliano; Puddu, Giovanna; Pujahari, Prabhat Ranjan; Punin, Valery; Putschke, Jorn Henning; Qvigstad, Henrik; Rachevski, Alexandre; Raha, Sibaji; Rajput, Sonia; Rak, Jan; Rakotozafindrabe, Andry Malala; Ramello, Luciano; Rami, Fouad; Raniwala, Rashmi; Raniwala, Sudhir; Rasanen, Sami Sakari; Rascanu, Bogdan Theodor; Rathee, Deepika; Ravasenga, Ivan; Read, Kenneth Francis; Redlich, Krzysztof; Reed, Rosi Jan; Rehman, Attiq Ur; Reichelt, Patrick Simon; Reidt, Felix; Ren, Xiaowen; Renfordt, Rainer Arno Ernst; Reolon, Anna Rita; Reshetin, Andrey; Reygers, Klaus Johannes; Riabov, Viktor; Ricci, Renato Angelo; Richert, Tuva Ora Herenui; Richter, Matthias Rudolph; Riedler, Petra; Riegler, Werner; Riggi, Francesco; Ristea, Catalin-Lucian; Rodriguez Cahuantzi, Mario; Rodriguez Manso, Alis; Roeed, Ketil; Rogochaya, Elena; Rohr, David Michael; Roehrich, Dieter; Ronchetti, Federico; Ronflette, Lucile; Rosnet, Philippe; Rossi, Andrea; Roukoutakis, Filimon; Roy, Ankhi; Roy, Christelle Sophie; Roy, Pradip Kumar; Rubio Montero, Antonio Juan; Rui, Rinaldo; Russo, Riccardo; Ryabinkin, Evgeny; Ryabov, Yury; Rybicki, Andrzej; Saarinen, Sampo; Sadhu, Samrangy; Sadovskiy, Sergey; Safarik, Karel; Sahlmuller, Baldo; Sahoo, Pragati; Sahoo, Raghunath; Sahoo, Sarita; Sahu, Pradip Kumar; Saini, Jogender; Sakai, Shingo; Saleh, Mohammad Ahmad; Salzwedel, Jai Samuel Nielsen; Sambyal, Sanjeev Singh; Samsonov, Vladimir; Sandor, Ladislav; Sandoval, Andres; Sano, Masato; Sarkar, Debojit; Sarkar, Nachiketa; Sarma, Pranjal; Scapparone, Eugenio; Scarlassara, Fernando; Schiaua, Claudiu Cornel; Schicker, Rainer Martin; Schmidt, Christian Joachim; Schmidt, Hans Rudolf; Schmidt, Martin; Schuchmann, Simone; Schukraft, Jurgen; Schutz, Yves Roland; Schwarz, Kilian Eberhard; Schweda, Kai Oliver; Scioli, Gilda; Scomparin, Enrico; Scott, Rebecca Michelle; Sefcik, Michal; Seger, Janet Elizabeth; Sekiguchi, Yuko; Sekihata, Daiki; Selyuzhenkov, Ilya; Senosi, Kgotlaesele; Senyukov, Serhiy; Serradilla Rodriguez, Eulogio; Sevcenco, Adrian; Shabanov, Arseniy; Shabetai, Alexandre; Shadura, Oksana; Shahoyan, Ruben; Shangaraev, Artem; Sharma, Ankita; Sharma, Mona; Sharma, Monika; Sharma, Natasha; Sheikh, Ashik Ikbal; Shigaki, Kenta; Shou, Qiye; Shtejer Diaz, Katherin; Sibiryak, Yury; Siddhanta, Sabyasachi; Sielewicz, Krzysztof Marek; Siemiarczuk, Teodor; Silvermyr, David Olle Rickard; Silvestre, Catherine Micaela; Simatovic, Goran; Simonetti, Giuseppe; Singaraju, Rama Narayana; Singh, Ranbir; Singhal, Vikas; Sarkar - Sinha, Tinku; Sitar, Branislav; Sitta, Mario; Skaali, Bernhard; Slupecki, Maciej; Smirnov, Nikolai; Snellings, Raimond; Snellman, Tomas Wilhelm; Song, Jihye; Song, Myunggeun; Song, Zixuan; Soramel, Francesca; Sorensen, Soren Pontoppidan; Sozzi, Federica; Spiriti, Eleuterio; Sputowska, Iwona Anna; Spyropoulou-Stassinaki, Martha; Stachel, Johanna; Stan, Ionel; Stankus, Paul; Stenlund, Evert Anders; Steyn, Gideon Francois; Stiller, Johannes Hendrik; Stocco, Diego; Strmen, Peter; Alarcon Do Passo Suaide, Alexandre; Sugitate, Toru; Suire, Christophe Pierre; Suleymanov, Mais Kazim Oglu; Suljic, Miljenko; Sultanov, Rishat; Sumbera, Michal; Sumowidagdo, Suharyo; Swain, Sagarika; Szabo, Alexander; Szarka, Imrich; Szczepankiewicz, Adam; Szymanski, Maciej Pawel; Tabassam, Uzma; Takahashi, Jun; Tambave, Ganesh Jagannath; Tanaka, Naoto; Tarhini, Mohamad; Tariq, Mohammad; Tarzila, Madalina-Gabriela; Tauro, Arturo; Tejeda Munoz, Guillermo; Telesca, Adriana; Terasaki, Kohei; Terrevoli, Cristina; Teyssier, Boris; Thaeder, Jochen Mathias; Thakur, Dhananjaya; Thomas, Deepa; Tieulent, Raphael Noel; Tikhonov, Anatoly; Timmins, Anthony Robert; Toia, Alberica; Trogolo, Stefano; Trombetta, Giuseppe; Trubnikov, Victor; Trzaska, Wladyslaw Henryk; Tsuji, Tomoya; Tumkin, Alexandr; Turrisi, Rosario; Tveter, Trine Spedstad; Ullaland, Kjetil; Uras, Antonio; Usai, Gianluca; Utrobicic, Antonija; Vala, Martin; Valencia Palomo, Lizardo; Van Der Maarel, Jasper; Van Hoorne, Jacobus Willem; Van Leeuwen, Marco; Vanat, Tomas; Vande Vyvre, Pierre; Varga, Dezso; Diozcora Vargas Trevino, Aurora; Vargyas, Marton; Varma, Raghava; Vasileiou, Maria; Vasiliev, Andrey; Vauthier, Astrid; Vazquez Doce, Oton; Vechernin, Vladimir; Veen, Annelies Marianne; Velure, Arild; Vercellin, Ermanno; Vergara Limon, Sergio; Vernet, Renaud; Vickovic, Linda; Viinikainen, Jussi Samuli; Vilakazi, Zabulon; Villalobos Baillie, Orlando; Villatoro Tello, Abraham; Vinogradov, Alexander; Vinogradov, Leonid; Virgili, Tiziano; Vislavicius, Vytautas; Viyogi, Yogendra; Vodopyanov, Alexander; Volkl, Martin Andreas; Voloshin, Kirill; Voloshin, Sergey; Volpe, Giacomo; Von Haller, Barthelemy; Vorobyev, Ivan; Vranic, Danilo; Vrlakova, Janka; Vulpescu, Bogdan; Wagner, Boris; Wagner, Jan; Wang, Hongkai; Wang, Mengliang; Watanabe, Daisuke; Watanabe, Yosuke; Weber, Michael; Weber, Steffen Georg; Weiser, Dennis Franz; Wessels, Johannes Peter; Westerhoff, Uwe; Whitehead, Andile Mothegi; Wiechula, Jens; Wikne, Jon; Wilk, Grzegorz Andrzej; Wilkinson, Jeremy John; Willems, Guido Alexander; Williams, Crispin; Windelband, Bernd Stefan; Winn, Michael Andreas; Yalcin, Serpil; Yang, Ping; Yano, Satoshi; Yin, Zhongbao; Yokoyama, Hiroki; Yoo, In-Kwon; Yoon, Jin Hee; Yurchenko, Volodymyr; Zaborowska, Anna; Zaccolo, Valentina; Zaman, Ali; Zampolli, Chiara; Correia Zanoli, Henrique Jose; Zaporozhets, Sergey; Zardoshti, Nima; Zarochentsev, Andrey; Zavada, Petr; Zavyalov, Nikolay; Zbroszczyk, Hanna Paulina; Zgura, Sorin Ion; Zhalov, Mikhail; Zhang, Haitao; Zhang, Xiaoming; Zhang, Yonghong; Chunhui, Zhang; Zhang, Zuman; Zhao, Chengxin; Zhigareva, Natalia; Zhou, Daicui; Zhou, You; Zhou, Zhuo; Zhu, Hongsheng; Zhu, Jianhui; Zichichi, Antonino; Zimmermann, Alice; Zimmermann, Markus Bernhard; Zinovjev, Gennady; Zyzak, Maksym

    2017-01-01

    At sufficiently high temperature and energy density, nuclear matter undergoes a transition to a phase in which quarks and gluons are not confined: the quark–gluon plasma (QGP). Such an exotic state of strongly interacting quantum chromodynamics matter is produced in the laboratory in heavy nuclei high-energy collisions, where an enhanced production of strange hadrons is observed. Strangeness enhancement, originally proposed as a signature of QGP formation in nuclear collisions, is more pronounced for multi-strange baryons. Several effects typical of heavy-ion phenomenology have been observed in high-multiplicity proton–proton (pp) collisions, but the enhanced production of multi-strange particles has not been reported so far. Here we present the first observation of strangeness enhancement in high-multiplicity proton–proton collisions. We find that the integrated yields of strange and multi-strange particles, relative to pions, increases significantly with the event charged-particle multiplicity. The me...

  1. Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients

    Science.gov (United States)

    Lahner, Edith; Brigatti, Cristina; Marzinotto, Ilaria; Carabotti, Marilia; Scalese, Giulia; Davidson, Howard W; Wenzlau, Janet M; Bosi, Emanuele; Piemonti, Lorenzo; Annibale, Bruno; Lampasona, Vito

    2017-01-01

    Objectives: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls. Methods: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies. Results: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (Pgastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa. PMID:28102858

  2. Precipitation in Zr-2.5Nb enhanced by proton irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Cann, C. D.; So, C. B.; Styles, R. C.; Coleman, C. E.

    1993-08-15

    A 3.6 MeV proton irradiation of annealed Zr-2.5Nb has been performed to determine whether proton irradiation will enhance the precipitation of Nb-rich {beta}-phase precipitates within the {alpha}-grains. a transmission electron microscope examination of a foil after irradiation at 770 K for 18 h and at 720 K for 264.5 h to a total damage of 0.94 dpa revealed a fine dispersion of precipitates within the {alpha}-grains. Electron diffraction analysis of the precipitates found they have lattice plane spacings consistent with the Nb-rich {beta}-phase. This result is in agreement with the {beta}phase precipitation observed following neutron irradiation, and thus it supports the use of proton irradiation to simulate neutron-irradiation effects in Zr-2.5Nb.

  3. Precipitation in Zr-2.5Nb enhanced by proton irradiation

    International Nuclear Information System (INIS)

    Cann, C.D.; So, C.B.; Styles, R.C.; Coleman, C.E.

    1993-08-01

    A 3.6 MeV proton irradiation of annealed Zr-2.5Nb has been performed to determine whether proton irradiation will enhance the precipitation of Nb-rich β-phase precipitates within the α-grains. a transmission electron microscope examination of a foil after irradiation at 770 K for 18 h and at 720 K for 264.5 h to a total damage of 0.94 dpa revealed a fine dispersion of precipitates within the α-grains. Electron diffraction analysis of the precipitates found they have lattice plane spacings consistent with the Nb-rich β-phase. This result is in agreement with the βphase precipitation observed following neutron irradiation, and thus it supports the use of proton irradiation to simulate neutron-irradiation effects in Zr-2.5Nb

  4. TU-H-CAMPUS-TeP3-03: Dose Enhancement by Gold Nanoparticles Around the Bragg Peak of Proton Beams

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, J; Sutherland, K [Department of Medical Physics, Hokkaido University Graduate School of Medicine (Japan); Hashimoto, T [Department of Radiation Medicine, Hokkaido University Graduate School of Medicine (Japan); Peng, H; Xing, L [Department of Radiation Oncology, Stanford University and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University (Japan); Shirato, H [Department of Radiation Medicine, Hokkaido University Graduate School of Medicine and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University (Japan); Date, H [Faculty of Health Sciences, Hokkaido University (Japan)

    2016-06-15

    Purpose: To make clear the spatial distribution of dose enhancement around gold nanoparticles (GNPs) located near the proton Bragg peak, and to evaluate the potential of GNPs as a radio sensitizer. Methods: The dose enhancement by electrons emitted from GNPs under proton irradiation was estimated by Geant4 Monte Carlo simulation toolkit in two steps. In an initial macroscopic step, 100 and 195 MeV proton beams were incident on a water cube, 30 cm on a side. Energy distributions of protons were calculated at four depths, 50% and 75% proximal to the Bragg peak, 100% peak, and 75% distal to the peak (P50, P75, Peak, and D75, respectively). In a subsequent microscopic step, protons with the energy distribution calculated above were incident on a 20 nm diameter GNP in a nanometer-size water box and the spatial distribution of dose around the GNP was determined for each energy distribution. The dose enhancement factor (DEF) was also deduced. Results: The dose enhancement effect was spread to several tens of nanometers in the both depth and radial directions. The enhancement area increased in the order of P50, P75, Peak, and D75 for both cases with 100 and 195 MeV protons. In every position around the Bragg peak, the 100 MeV beam resulted in a higher dose enhancement than the 195 MeV beam. At P75, the average and maximum DEF were 3.9 and 17.0 for 100 MeV, and 3.5 and 16.2 for 195 MeV, respectively. These results indicate that lower energy protons caused higher dose enhancement in this incident proton energy range. Conclusion: The dose enhancement around GNPs spread as the position in the Bragg peak region becomes deeper and depends on proton energy. It is expected that GNPs can be used as a radio sensitizer with consideration of the location and proton beam energy.

  5. Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice1

    Science.gov (United States)

    Jimenez, Tamara; Sanchez, Gladis; McDermott, Jeffrey P.; Nguyen, Anh-Nguyet; Kumar, T. Rajendra; Blanco, Gustavo

    2010-01-01

    The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions. PMID:20826726

  6. Identification of V-ATPase as a molecular sensor of SOX11-levels and potential therapeutic target for mantle cell lymphoma

    International Nuclear Information System (INIS)

    Emruli, Venera Kuci; Olsson, Roger; Ek, Fredrik; Ek, Sara

    2016-01-01

    Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/β-catenin pathway that is of importance for proliferation in MCL. Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation. An inducible SOX11 knock-down system was used to functionally screen a library of compounds (n = 75) targeting the Wnt signaling pathway. A functionally interesting target, vacuolar-type H + -ATPase (V-ATPase), was further evaluated by western blot, siRNA-mediated gene silencing, immunofluorescence, and flow cytometry. We show that 15 out of 75 compounds targeting the Wnt pathway reduce proliferation in all three MCL cell lines tested. Furthermore, three substances targeting two different targets (V-ATPase and Dkk1) showed SOX11-dependent activity. Further validation analyses were focused on V-ATPase and showed that two independent V-ATPase inhibitors (bafilomycin A1 and concanamycin A) are sensitive to SOX11 levels, causing reduced anti-proliferative response in SOX11 low cells. We further show, using fluorescence imaging and flow cytometry, that V-ATPase is mainly localized to the plasma membrane in primary and MCL cell lines. We show that SOX11 status affect V-ATPase dependent pathways, and thus may be involved in regulating pH in intracellular and extracellular compartments. The plasma membrane localization of V-ATPase indicates that pH regulation of the immediate extracellular compartment may be of importance for receptor functionality and potentially invasiveness in vivo. The online version of this article (doi:10

  7. Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

    DEFF Research Database (Denmark)

    Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

    2007-01-01

    In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na(+),K(+)-ATPase and the sarco(endo)plasmic reticulum Ca(2+)-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported...... cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca(2+) interaction/occlusion in Ca(2+)-ATPase and K(+) interaction/occlusion in Na(+),K(+)-ATPase. Leu(65) of the Ca(2+)-ATPase and Leu(99) of the Na(+),K(+)-ATPase, located...... of the extracytoplasmic gate in both the Ca(2+)-ATPase and the Na(+),K(+)-ATPase. Udgivelsesdato: 2007-Dec...

  8. Enhancement of proton acceleration field in laser double-layer target interaction

    International Nuclear Information System (INIS)

    Gu, Y. J.; Kong, Q.; Li, X. F.; Yu, Q.; Wang, P. X.; Kawata, S.; Izumiyama, T.; Ma, Y. Y.

    2013-01-01

    A mechanism is proposed to enhance a proton acceleration field in laser plasma interaction. A double-layer plasma with different densities is illuminated by an intense short pulse. Electrons are accelerated to a high energy in the first layer by the wakefield. The electrons accelerated by the laser wakefield induce the enhanced target normal sheath (TNSA) and breakout afterburner (BOA) accelerations through the second layer. The maximum proton energy reaches about 1 GeV, and the total charge with an energy higher than 100 MeV is about several tens of μC/μm. Both the acceleration gradient and laser energy transfer efficiency are higher than those in single-target-based TNSA or BOA. The model has been verified by 2.5D-PIC simulations

  9. Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

    International Nuclear Information System (INIS)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-01-01

    Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

  10. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  11. Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase

    International Nuclear Information System (INIS)

    Stewart, J.M.; Jorgensen, P.L.; Grisham, C.M.

    1989-01-01

    Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound (Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme

  12. Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Stewart, J.M.; Jorgensen, P.L.; Grisham, C.M. (Univ. of Virginia, Charlottesville (USA))

    1989-05-30

    Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound (Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35{degrees}, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.

  13. Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions.

    Science.gov (United States)

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

  14. SU-F-T-666: Molecular-Targeted Gold Nanorods Enhances the RBE of Proton Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Khoo, A; Sahoo, N; Krishnan, S; Diagaradjane, P [UT MD Anderson Cancer Center, Houston, TX (United States)

    2016-06-15

    Purpose: In recent years, proton beam radiation therapy (PBRT) has gained significant attention in the treatment of tumors in anatomically complex locations. However, the therapeutic benefit of PBRT is limited by a relative biological effectiveness (RBE) of just 1.1. The purpose of this study is to evaluate whether this limitation can be overcome by artificially enhancing the RBE using molecular-targeted gold nanorods (GNRs). Methods: Molecular-targeting of GNRs was accomplished using Cetuximab (antibody specific to epidermal growth factor receptor that is over-expressed in tumors) conjugated GNRs (cGNRs) and their binding affinity to Head and Neck cancer cells was confirmed using dark field microscopy and Transmission Electron Microscopy (TEM). The radiosensitization potential of cGNRs when irradiated with photon (6MV) and proton (100 and 160 MeV) beams was determined using clonogenic assays. The RBE at 10% surviving fraction (RBE{sub 10}) for proton therapies at central and distal locations of SOBP was calculated with respect to 6 MV photons. IgGconjugated GNRs (iGNRs) were used as controls in all experiments. Results: cGNRs demonstrated significant radiosensitization when compared to iGNRs for 6MV photons (1.14 vs 1.04), 100 MeV protons (1.19 vs 1.04), and 160 MeV protons (1.17 vs 1.04). While RBE10 for proton beams at the center of SOBP revealed similar effects for both 100 and 160 MeV (RBE{sup 10}=1.39 vs 1.38; p>0.05), enhanced radiosensitization was observed at the distal SOBP with 100 MeV beams demonstrating greater effect than 160 MeV beams (RBE{sup 10}=1.79 vs 1.6; p<0.05). Conclusion: EGFR-targeting GNRs significantly enhance the RBE of protons well above the accepted 1.1 value. The enhanced RBE observed for lower energy protons (100 MeV) and at the distal SOBP suggests that low energy components may play a role in the observed radiosensitization effect. This strategy holds promise for clinical translation and could evolve as a paradigm-changing approach

  15. -pumping pyrophosphatase in pepper plants

    KAUST Repository

    Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Gregoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

    2018-01-01

    It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved.

  16. -pumping pyrophosphatase in pepper plants

    KAUST Repository

    Vigani, Gianpiero

    2018-05-22

    It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves\\' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved.

  17. Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

    DEFF Research Database (Denmark)

    Axelsen, K.B.; Venema, K.; Jah, T.

    1999-01-01

    in an extension of the C-terminus unique to plant H+-ATPases, Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain......The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues...

  18. Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

    Science.gov (United States)

    Schmid, T. E.; Greubel, C.; Hable, V.; Zlobinskaya, O.; Michalski, D.; Girst, S.; Siebenwirth, C.; Schmid, E.; Molls, M.; Multhoff, G.; Dollinger, G.

    2012-10-01

    This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm-1) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBEMN = 1.48 ± 0.07) and dicentrics (RBED = 1.92 ± 0.15), in human-hamster hybrid (AL) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm2 matrix compared to quasi homogeneous in a 1 × 1 µm2 matrix applied protons (RBEMN = 1.28 ± 0.07; RBED = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a 12C ion with 55 MeV total energy (4.48 MeV u-1). The enhancements are about half of that obtained for 12C ions (RBEMN = 2.20 ± 0.06 and RBED = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.

  19. Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter.

    Science.gov (United States)

    Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro; Krulwich, Terry Ann

    2014-01-01

    Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).

  20. Interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-ATPase localization and function.

    Science.gov (United States)

    Wee, Yin Shen; Roundy, Kirstin M; Weis, Janis J; Weis, John H

    2012-12-01

    The innate response interferon-inducible transmembrane (Ifitm) proteins have been characterized as influencing proliferation, signaling complexes and restricting virus infections. Treatment of cells lacking these proteins (IfitmDel) with IFN-β resulted in the loss of clathrin from membrane compartments and the inhibition of clathrin-mediated phagocytosis, suggesting a molecular interaction between clathrin and Ifitm proteins. The pH of endosomes of IfitmDel cells, with or without IFN activation, was neutralized, suggesting the function of the vacular ATPase proton pumps in such cells was compromised. Co-immunoprecipitation of Ifitm3 with Atp6v0b demonstrated a direct interaction between the Ifitm proteins and the v-ATPase. These data suggest that the Ifitm proteins help stabilize v-ATPase complexes in cellular membranes which, in turn, facilitates the appropriate subcellular localization of clathrin.

  1. Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase1[OPEN

    Science.gov (United States)

    Tan, Li Xuan; Bushey, Daniel B.; Swanson, Sarah J.

    2018-01-01

    A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase. PMID:29042459

  2. The Role of the Plasma Membrane H+-ATPase in Plant Responses to Aluminum Toxicity

    Directory of Open Access Journals (Sweden)

    Jiarong Zhang

    2017-10-01

    Full Text Available Aluminum (Al toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H+-ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H+-ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H+-ATPase mediated H+ influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H+-ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H+-ATPase through application of its activators (e.g., magnesium or IAA or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H+-ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

  3. Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis.

    Science.gov (United States)

    Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

    2011-07-01

    Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  4. On archaebacterial ATPase from Halobacterium saccharovorum

    Science.gov (United States)

    Kristjansson, H.; Ponnamperuma, C.; Hochstein, L.; Altekar, W.

    1984-01-01

    The energy transducing ATPase from Halobacterium saccharovorum was studied in order to define the origin of energy transducing systems. The ATPase required high salt concentration (4M NaCl) for activity; activity was rapidly lost when NaCl was below 1 Molar. At low salt concentration, the membrane bound ATPase activity could be stabilized in presence of spermine. However, following solubilization spermine was ineffective. Furthermore, F1 ATPase activity was stabilized by ammonium sulfate even when the NaCl concentration was less than 1 Molar. These studies suggest that stabilization by hydrophobic interactions preceded ionic ones in the evolution of the energy transducing ATPases.

  5. Cell potentials, cell resistance, and proton fluxes in corn root tissue. Effects of dithioerythritol

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W.; Hanson, J.B.

    1976-09-01

    Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H/sup +/ and K/sup +/ fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents. We deduce that dithioerythritol activates a passive H/sup +//K/sup +/ antiport, driven in these experiments by the outwardly directed electrochemical gradient of K/sup +/. The net effect on H/sup +/ and K/sup +/ fluxes is believed to reside with the combined activity of a polarized H/sup +//K/sup +/ exchanging ATPase and the passive H/sup +//K/sup +/ antiport. A model is presented to show how the combined system might produce stable potential differences and K/sup +/ content.

  6. Stimulation of Na+/K+ ATPase activity and Na+ coupled glucose transport by β-catenin

    International Nuclear Information System (INIS)

    Sopjani, Mentor; Alesutan, Ioana; Wilmes, Jan; Dermaku-Sopjani, Miribane; Lam, Rebecca S.; Koutsouki, Evgenia; Jakupi, Muharrem; Foeller, Michael; Lang, Florian

    2010-01-01

    Research highlights: → The oncogenic transcription factor β-catenin stimulates the Na + /K + -ATPase. → β-Catenin stimulates SGLT1 dependent Na + , glucose cotransport. → The effects are independent of transcription. → β-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: β-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. β-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that β-catenin influences membrane transport. To this end, β-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of β-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na + /K + -ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of β-catenin on the endogenous Na + /K + -ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of β-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of β-catenin expression. The stimulating effect of β-catenin on both Na + /K + ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of β-catenin, i.e. the regulation of transport.

  7. Active transport of Na/sup +/ by reconstituted Na,K-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Boldyrev, A.A.; Svinukhova, I.A.

    1987-02-20

    The ability of ATP, CTP, ITP, GTP, and UTP to support ouabain-sensitive accumulation of Na/sup +/ by proteoliposomes with a reconstituted Na/K-pump was investigated. At a low (Na/sup +/)/(K/sup +/) ratio in the medium (20 mM/50 mM), a correlation is observed between the proton-accepting capacity of the nucleotide and its effectiveness as a substrate of active transport. To test the hypothesis of the importance of the presence of a negative charge in the 1-position of the purine (3-pyrimidine) base of the nucleotide for mutual transitions between the Na- and K-conformations of Na,K-ATPase they used two analogs of ATP: N/sub 1/-hydroxy-ATP, possessing proton acceptor capacity, and N/sub 1/-methoxy-ATP, in the molecule of which the negative charge is quenched by a methyl group. The first substrate supports active accumulation of Na/sup +/ in proteoliposomes at the same rate as ATP, whereas the second substrate is relatively ineffective.

  8. Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

    International Nuclear Information System (INIS)

    Schmid, T E; Zlobinskaya, O; Michalski, D; Molls, M; Multhoff, G; Greubel, C; Hable, V; Girst, S; Siebenwirth, C; Dollinger, G; Schmid, E

    2012-01-01

    This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm −1 ) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBE MN = 1.48 ± 0.07) and dicentrics (RBE D = 1.92 ± 0.15), in human–hamster hybrid (A L ) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm 2 matrix compared to quasi homogeneous in a 1 × 1 µm 2 matrix applied protons (RBE MN = 1.28 ± 0.07; RBE D = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a 12 C ion with 55 MeV total energy (4.48 MeV u −1 ). The enhancements are about half of that obtained for 12 C ions (RBE MN = 2.20 ± 0.06 and RBE D = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles. (paper)

  9. Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition

    Directory of Open Access Journals (Sweden)

    Antônio Jesus Dorighetto Cogo

    2018-02-01

    Full Text Available Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

  10. Proton detection for signal enhancement in solid-state NMR experiments on mobile species in membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ward, Meaghan E.; Ritz, Emily [University of Guelph, Department of Physics (Canada); Ahmed, Mumdooh A. M. [Suez University, The Department of Physics, Faculty of Science (Egypt); Bamm, Vladimir V.; Harauz, George [University of Guelph, Biophysics Interdepartmental Group (Canada); Brown, Leonid S.; Ladizhansky, Vladimir, E-mail: vladizha@uoguelph.ca [University of Guelph, Department of Physics (Canada)

    2015-12-15

    Direct proton detection is becoming an increasingly popular method for enhancing sensitivity in solid-state nuclear magnetic resonance spectroscopy. Generally, these experiments require extensive deuteration of the protein, fast magic angle spinning (MAS), or a combination of both. Here, we implement direct proton detection to selectively observe the mobile entities in fully-protonated membrane proteins at moderate MAS frequencies. We demonstrate this method on two proteins that exhibit different motional regimes. Myelin basic protein is an intrinsically-disordered, peripherally membrane-associated protein that is highly flexible, whereas Anabaena sensory rhodopsin is composed of seven rigid transmembrane α-helices connected by mobile loop regions. In both cases, we observe narrow proton linewidths and, on average, a 10× increase in sensitivity in 2D insensitive nuclear enhancement of polarization transfer-based HSQC experiments when proton detection is compared to carbon detection. We further show that our proton-detected experiments can be easily extended to three dimensions and used to build complete amino acid systems, including sidechain protons, and obtain inter-residue correlations. Additionally, we detect signals which do not correspond to amino acids, but rather to lipids and/or carbohydrates which interact strongly with membrane proteins.

  11. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  12. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid; Rapoport, Tom A. (UMASS, MED); (Harvard-Med)

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  13. Translocation, switching and gating: potential roles for ATP in long-range communication on DNA by Type III restriction endonucleases.

    Science.gov (United States)

    Szczelkun, Mark D

    2011-04-01

    To cleave DNA, the Type III RM (restriction-modification) enzymes must communicate the relative orientation of two recognition sequences, which may be separated by many thousands of base pairs. This long-range interaction requires ATP hydrolysis by a helicase domain, and both active (DNA translocation) and passive (DNA sliding) modes of motion along DNA have been proposed. Potential roles for ATP binding and hydrolysis by the helicase domains are discussed, with a focus on bipartite ATPases that act as molecular switches.

  14. 1H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla

    International Nuclear Information System (INIS)

    Stewart, J.M.M.; Grisham, C.M.

    1988-01-01

    1 H nuclear magnetic relaxation measurements have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH 3 ) 4 ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH 4 ) 4 ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase and that Mn 2+ bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na-K-ATPase. From the paramagnetic effect of Mn 2+ bound to the APTase on the longitudinal relaxation rates of the protons of Co(NH 3 ) 4 ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous 31 P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35 0 , and the conformation of the ribose ring is slightly N-type. The bound Mn 2+ lies above and in the plane of the adenine ring. The distances from Mn 2+ to N 6 and N 7 are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme

  15. A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform.

    Science.gov (United States)

    Hirz, Melanie; Richter, Gerald; Leitner, Erich; Wriessnegger, Tamara; Pichler, Harald

    2013-11-01

    The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure-function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [(3)H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.

  16. Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na+,K+-ATPase inhibitors in acutely volume-expanded hog

    International Nuclear Information System (INIS)

    Tamura, M.; Harris, T.M.; Higashimori, K.; Sweetman, B.J.; Blair, I.A.; Inagami, T.

    1987-01-01

    Na + ,K + -ATPase inhibitors activities against the specific binding of ouabain to Na + ,K + -ATPase and 86 Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as γ-arachidoyl- [LPCA(γ), 34%], β-arachidoyl- [LPCA(β), 4%], γ-linoleoyl- (LPCL, 33%), and γ-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of γ-docosapentaenoyl-, γ-eicosatrienoyl-, and γpalmitoyllysophosphatidylcholine were also detected by both FAB mass and 1 H NMR spectrometric studies. The inhibition of Na + ,K + -ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and 86 Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest that γ-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na + ,K + -ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme

  17. Comprehensive comparison of two protein family of P-ATPases (13A1 and 13A3) in insects.

    Science.gov (United States)

    Seddigh, Samin

    2017-06-01

    The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms. Copyright © 2017 Elsevier Ltd. All rights

  18. Proton tunneling-induced bistability, oscillations and enhanced performance of PEM fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Katsaounis, A.; Balomenou, S.; Tsiplakides, D.; Brosda, S.; Vayenas, C.G. [Department of Chemical Engineering, University of Patras, Patras GR 26504 (Greece); Neophytides, S. [Institute of Chemical Engineering and High Temperature Chemical Processes, FORTH, 26500 Patras (Greece)

    2005-03-25

    Proton migration through hydrated Nafion membranes in polymer electrolyte membrane (PEM) fuel cells occurs both in the aqueous phase of the membrane and on the sulfonate groups on the surface of the membrane pores. Here we show using D{sub 2} and H{sub 2} fuel and basic quantum mechanical equations that this surface proton migration is largely due to proton tunneling between adjacent sulfonate groups, leading to an exponential variation of Nafion conductivity with cell potential. This amphibious mode of proton migration, particle-like in the aqueous phase and wave-like in the narrow pores, is shown to be the major cause of cell overpotential, bistability and oscillations of state-of-the-art PEM fuel cells operating on H{sub 2}, reformate or methanol fuel. We also show that this phenomenon can be exploited via introduction of a third auxiliary electrode to independently control the anode-cathode potential difference and dramatically enhance fuel cell power output even in absence of noble metals at the anode.

  19. A novel heavy metal ATPase peptide from Prosopis juliflora is involved in metal uptake in yeast and tobacco.

    Science.gov (United States)

    Keeran, Nisha S; Ganesan, G; Parida, Ajay K

    2017-04-01

    Heavy metal pollution of agricultural soils is one of the most severe ecological problems in the world. Prosopis juliflora, a phreatophytic tree species, grows well in heavy metal laden industrial sites and is known to accumulate heavy metals. Heavy Metal ATPases (HMAs) are ATP driven heavy metal pumps that translocate heavy metals across biological membranes thus helping the plant in heavy metal tolerance and phytoremediation. In the present study we have isolated and characterized a novel 28.9 kDa heavy metal ATPase peptide (PjHMT) from P. juliflora which shows high similarity to the C-terminal region of P 1B ATPase HMA1. It also shows the absence of the invariant signature sequence DKTGT, and the metal binding CPX motif but the presence of conserved regions like MVGEGINDAPAL (ATP binding consensus sequence), HEGGTLLVCLNS (metal binding domain) and MLTGD, GEGIND and HEGG motifs which play important roles in metal transport or ATP binding. PjHMT, was found to be upregulated under cadmium and zinc stress. Heterologous expression of PjHMT in yeast showed a higher accumulation and tolerance of heavy metals in yeast. Further, transgenic tobacco plants constitutively expressing PjHMT also showed increased accumulation and tolerance to cadmium. Thus, this study suggests that the transport peptide from P. juliflora may have an important role in Cd uptake and thus in phytoremediation.

  20. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

    Science.gov (United States)

    The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1– expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and p...

  2. Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress.

    Science.gov (United States)

    Aslam, Roohi; Williams, Lorraine E; Bhatti, Muhammad Faraz; Virk, Nasar

    2017-10-27

    P 2 - type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca 2+ , Mn 2+ and Zn 2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat. In this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P 2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P 2- type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat. Here we concluded that wheat genome consists of nine P 2B and three P 2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P 2A and P 2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be

  3. Calculating the Na⁺ translocating V-ATPase catalytic site affinity for substrate binding by homology modeled NtpA monomer using molecular dynamics/free energy calculation.

    Science.gov (United States)

    Muhammed, Zahed; Arai, Satoshi; Saijo, Shinya; Yamato, Ichiro; Murata, Takeshi; Suenaga, Atsushi

    2012-07-01

    Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and two peripheral stalks (NtpC, NtpD-G and NtpE-F). Recently nucleotide binding of catalytic NtpA monomer has been reported (Arai et al.). In the present study, we calculated the nucleotide binding affinity of NtpA by molecular dynamics (MD) simulation/free energy calculation using MM-GBSA approach based on homology modeled structure of NtpA monomer docked with ATP analogue, adenosine 5'-[β, γ-imido] triphosphate (AMP-PNP). The calculated binding free energies showed qualitatively good agreement with experimental data. The calculation was cross-validated further by the rigorous method, thermodynamic integration (TI) simulation. Finally, the interaction between NtpA and nucleotides at the atomic level was investigated by the analyses of components of free energy and the optimized model structures obtained from MD simulations, suggesting that electrostatic contribution is responsible for the difference in nucleotide binding to NtpA monomer. This is the first observation and suggestion to explain the difference of nucleotide binding properties in V-ATPase NtpA subunit, and our method can be a valuable primary step to predict nucleotide binding affinity to other subunits (NtpAB, NtpA₃B₃) and to explore subunit interactions and eventually may help to understand energy transduction mechanism of E. hirae V-ATPase. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. The role of endomembrane-localized VHA-c in plant growth.

    Science.gov (United States)

    Zhou, Aimin; Takano, Tetsuo; Liu, Shenkui

    2018-01-02

    In plant cells, the vacuolar-type H + -ATPase (V-ATPase), a large multis`ubunit endomembrane proton pump, plays an important role in acidification of subcellular organelles, pH and ion homeostasis, and endocytic and secretory trafficking. V-ATPase subunit c (VHA-c) is essential for V-ATPase assembly, and is directly responsible for binding and transmembrane transport of protons. In previous studies, we identified a PutVHA-c gene from Puccinellia tenuiflora, and investigated its function in plant growth. Subcellular localization revealed that PutVHA-c is mainly localized in endosomal compartments. Overexpression of PutVHA-c enhanced V-ATPase activity and promoted plant growth in transgenic Arabidopsis. Furthermore, the activity of V-ATPase affected intracellular transport of the Golgi-derived endosomes. Our results showed that endomembrane localized-VHA-c contributes to plant growth by influencing V-ATPase-dependent endosomal trafficking. Here, we discuss these recent findings and speculate on the VHA-c mediated molecular mechanisms involved in plant growth, providing a better understanding of the functions of VHA-c and V-ATPase.

  5. MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

    International Nuclear Information System (INIS)

    Milgrom, Elena M.; Milgrom, Yakov M.

    2012-01-01

    Highlights: ► MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. ► V-ATPase activity saturation with MgATP is not sufficient for complete protection. ► The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K m values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

  6. FXYD5: Na+/ K+-ATPase Regulator in Health and Disease.

    Directory of Open Access Journals (Sweden)

    Irina eLubarski Gotliv

    2016-03-01

    Full Text Available FXYD5 (Dysadherin, RIC is a single span type I membrane protein that plays multiple roles in regulation of cellular functions. It is expressed in a variety of epithelial tissues and acts as an auxiliary subunit of the Na+/ K+-ATPase. During the past decade, a correlation between enhanced expression of FXYD5 and tumor progression has been established for various tumor types. In this review, current knowledge on FXYD5 is discussed, including experimental data on the functional effects of FXYD5 on the Na+/ K+-ATPase. FXYD5 modulates cellular junctions, influences chemokine production, and affects cell adhesion. The accumulated data may provide a basis for understanding the molecular mechanisms underlying FXYD5 mediated phenotypes.

  7. Influence of nitric acid synthesis on the function of (Na, K)-ATPase in the heart

    International Nuclear Information System (INIS)

    Vrbjar, N.; Bernathova, I.; Pechanova, O.

    1998-01-01

    Function of the sodium pump was characterized by kinetic parameters of the (Na,K)-ATPase at normal and lower synthesis of nitric oxide (NO) in the rat heart. Our findings indicate that there is no change in energy utilization by the cardiac sodium pump during lowered NO-synthesis. The transport properties of the enzyme are deteriorated, due to its decreased sensitivity to Na + . Inhibition of NO-synthesis in acute experiment by high doses of L-arginine analogue decreased the activity of (Na,K)-ATPase [Biol. Neonate, 68, 419 (1995)], an enzyme involved in the active translocation of Na + and K + ions across cell membranes. The present study was designed to investigate the influence of chronic inhibition of NO-synthesis on function of Na,K)-ATPase. One group (n=8) of adult male Wistar rats served as controls and the second group (n=16+ was treated with L-arginine analogue, the N G -nitro-L-arginine methyl ester (L-NAME) in a dose 40 mg/kg/day in drinking water for 4 weeks. NO-synthase activity was determined in crude homogenates of cardiac tissue by measuring the production of [ 3 H]-L-citrulline from from [ 3 H]-L-arginine. Chronic administration of L-NAME induced a significant inhibition (to 30%) of the NO-synthase activity. In control group the activity of NO-synthase amounted 11.44 ± 1.03 and in the L-NAME group it was 3.19 ± pmol L-Cit/min/mg protein. At the activation with ATP the shortage in NO-synthesis induced by L-NAME did not provoke significant changes in both, the V max and the K m value. This indicates that during the lower synthesis of NO there is no change in energy consumption for the transport of Na + and K + ions by the (Na,K)-ATPase. The stable value of K m indicates that shortage in NO does not induce changes in the vicinity of the ATP-binding site. On the other hand our results indicate that at lowered NO-synthesis the (Na,K)-ATPase in myocardium changes its Na + -binding and probably also the Na + -transport properties as it revealed from the

  8. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Directory of Open Access Journals (Sweden)

    Prabhanshu Kumar

    2014-10-01

    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  9. Impact of Gastric H+/K+-ATPase rs2733743 on the Intragastric pH-Values of Dexlansoprazole Injection in Chinese Subjects

    Directory of Open Access Journals (Sweden)

    Lu-Ning Sun

    2017-09-01

    Full Text Available Background: Not all patients with acid-related disorders receiving proton pump inhibitor (PP treatment get adequate gastric pH control. The genetic variation of receptors, metabolic enzymes, and transporters are known to cause failures of therapies. We have conducted a study to evaluate the influence of gastric H+/K+-ATPase, CYP2C19, and ABCB1 polymorphisms on the pharmacokinetic and pharmacodynamic profiles of dexlansoprazole injection in healthy Chinese subjects.Methods: A total of 51 subjects were enrolled for pharmacokinetic and pharmacodynamic study after a single intravenous administration of 20 or 30 mg dexlansoprazole. Plasma concentrations were determined using a chiral liquid chromatography-mass spectrometry method. The intragastric pH and baseline-adjusted intragastric pH parameters were introduced to evaluate the pharmacodynamic characters. Genotyping was performed by polymerase chain reaction.Results: The pharmacokinetic parameters were significantly influenced by CYP2C19 phenotypes, and gastric acid secretion inhibition were affected by both gastric H+/K+-ATPase and CYP2C19 polymorphisms. Gastric H+/K+-ATPase genotypes had greater effects than CYP2C19 genotypes on the suppression of gastric acid secretion.Conclusion: Gastric H+/K+-ATPase polymorphism may be one of the main reasons that cause insufficient gastric acid inhibition.

  10. Restrained Proton Indicator in Combined Quantum-Mechanics/Molecular-Mechanics Dynamics Simulations of Proton Transfer through a Carbon Nanotube.

    Science.gov (United States)

    Duster, Adam W; Lin, Hai

    2017-09-14

    Recently, a collective variable "proton indicator" was purposed for tracking an excess proton solvated in bulk water in molecular dynamics simulations. In this work, we demonstrate the feasibility of utilizing the position of this proton indicator as a reaction coordinate to model an excess proton migrating through a hydrophobic carbon nanotube in combined quantum-mechanics/molecular-mechanics simulations. Our results indicate that applying a harmonic restraint to the proton indicator in the bulk solvent near the nanotube pore entrance leads to the recruitment of water molecules into the pore. This is consistent with an earlier study that employed a multistate empirical valence bond potential and a different representation (center of excess charge) of the proton. We attribute this water recruitment to the delocalized nature of the solvated proton, which prefers to be in high-dielectric bulk solvent. While water recruitment into the pore is considered an artifact in the present simulations (because of the artificially imposed restraint on the proton), if the proton were naturally restrained, it could assist in building water wires prior to proton transfer through the pore. The potential of mean force for a proton translocation through the water-filled pore was computed by umbrella sampling, where the bias potentials were applied to the proton indicator. The free energy curve and barrier heights agree reasonably with those in the literature. The results suggest that the proton indicator can be used as a reaction coordinate in simulations of proton transport in confined environments.

  11. Novel aspects of Na+,K+-ATPase

    OpenAIRE

    Aizman, Oleg

    2002-01-01

    Na,K-ATPase, an integral membrane protein expressed in each eukaryotic cell, serves as the major determinant of intracellular ion composition. In the current study we investigated novel aspects of Na,K-ATPase function and regulation. It is well established that Na,K-ATPase activity is regulated by reversible phosphorylation. New findings in this study are: 1) the level of intracellular Ca 2. concentration determines the functional effects of PKA and PKC-mediated Na,K-ATP...

  12. The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.

    NARCIS (Netherlands)

    Pont, J.J.H.H.M. de; Swarts, H.G.P.; Karawajczyk, A.; Schaftenaar, G.; Willems, P.H.G.M.; Koenderink, J.B.

    2009-01-01

    Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of

  13. Tissue-specific Role of the Na,K-ATPase α2 Isozyme in Skeletal Muscle*

    Science.gov (United States)

    Radzyukevich, Tatiana L.; Neumann, Jonathon C.; Rindler, Tara N.; Oshiro, Naomi; Goldhamer, David J.; Lingrel, Jerry B.; Heiny, Judith A.

    2013-01-01

    The Na,K-ATPase α2 isozyme is the major Na,K-ATPase of mammalian skeletal muscle. This distribution is unique compared with most other cells, which express mainly the Na,K-ATPase α1 isoform, but its functional significance is not known. We developed a gene-targeted mouse (skα2−/−) in which the α2 gene (Atp1a2) is knocked out in the skeletal muscles, and examined the consequences for exercise performance, membrane potentials, contractility, and muscle fatigue. Targeted knockout was confirmed by genotyping, Western blot, and immunohistochemistry. Skeletal muscle cells of skα2−/− mice completely lack α2 protein and have no α2 in the transverse tubules, where its expression is normally enhanced. The α1 isoform, which is normally enhanced on the outer sarcolemma, is up-regulated 2.5-fold without change in subcellular targeting. skα2−/− mice are apparently normal under basal conditions but show significantly reduced exercise capacity when challenged to run. Their skeletal muscles produce less force, are unable to increase force to match demand, and show significantly increased susceptibility to fatigue. The impairments affect both fast and slow muscle types. The subcellular targeting of α2 to the transverse tubules is important for this role. Increasing Na,K-ATPase α1 content cannot fully compensate for the loss of α2. The increased fatigability of skα2−/− muscles is reproduced in control extensor digitorum longus muscles by selectively inhibiting α2 enzyme activity with ouabain. These results demonstrate that the Na,K-ATPase α2 isoform performs an acute, isoform-specific role in skeletal muscle. Its activity is regulated by muscle use and enables working muscles to maintain contraction and resist fatigue. PMID:23192345

  14. Mg,Ca-ATPase activity under irradiation

    International Nuclear Information System (INIS)

    Ladutin, V.V.; Orlova, V.V.; Lob, P.A.; Gerasiminko, I.V.; Mack, E.I.

    2003-01-01

    Full text: The influence of different doses irradiation at the Mg,Ca-ATPase activity at the rat brain has been investigated. The analyses were made at the apparatus of LKB and Carl-Ceis-Jena firm with help of reagents of Sigma and Boehringer firm. Rats decapitated after 1, 3, 6, 24 and 48 h after action of irradiation. Dose 0.206 C/kg. Erythrocytes. 1 and 3h after irradiation influence- decrease of Mg,Ca-ATPase activity to 86-87% relatively control level, 24 and 48 h - increase of activity to the control level. Dose 0.312 C/kg. Large hemispheres. 1h - decrease of ATPase activity to 90% relatively control, 3h - increase to control level, 24h - fall to 86%, after 48h small increase to 93% relatively control. Dose 9.287 C/kg. Large hemispheres. 1h - sharp fall of Mg, Ca-ATPase activity to 67 % relatively control, increase of activity to 96% after 3h and sharp fall of activity to 64% 6h after action of irradiation. Dose 9.287 C/kg. Cerebellum. 1h - sharp decrease of ATPase activity to 80%. After 3h -sharp increase to 160% relatively control level and sharp fall of ATPase activity to 47% relatively control after 6h. The mechanism of radiation pathology of active ion transport has been discussed

  15. Identification of Na+/K+-ATPase inhibitors in bovine plasma as fatty acids and hydrocarbons

    DEFF Research Database (Denmark)

    Tal, D M; Yanuck, M D; Van Hall, Gerrit

    1989-01-01

    A preparative purification of endogenous inhibitors of the Na+/K+-ATPase has been carried out from bovine blood. Dried plasma was deproteinized, hexane-extracted and desalted, followed by further purification through a series of reverse-phase HPLC fractionations. Fractions active in inhibiting Na...... ouabain, and in addition it enhanced ouabain binding at high dilutions. These properties are indicative of nonspecific interactions with the Na+/K+-ATPase. The active fraction was identified by TLC, HPLC, NMR, GLC and GC-MS, to be a mixture of three unesterified fatty acids, mainly oleic acid (72...

  16. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3' to 5' translocase and helicase activities.

    Science.gov (United States)

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-08-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and a C-terminal DUF1998 domain (containing a putative tetracysteine metal-binding motif). We show that SftH is a monomeric DNA-dependent ATPase/dATPase that translocates 3' to 5' on single-stranded DNA and has 3' to 5' helicase activity. SftH homologs are found in bacteria representing 12 different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis). SftH homologs are evident in more than 30 genera of Archaea. Among eukarya, SftH homologs are present in plants and fungi.

  17. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

    Science.gov (United States)

    Oliver, A. E.; Deamer, D. W.

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two

  18. Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca exchangers.

    Science.gov (United States)

    Barkla, Bronwyn J; Hirschi, Kendal D; Pittman, Jon K

    2008-05-01

    Tonoplast-localised proton-coupled Ca(2+) transporters encoded by cation/H(+)exchanger (CAX) genes play a critical role in sequestering Ca(2+) into the vacuole. These transporters may function in coordination with Ca(2+) release channels, to shape stimulus-induced cytosolic Ca(2+) elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant's stress response. A common feature of these mutants was the perturbation of H(+)-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca(2+)/H(+) exchangers and H(+) pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca(2+) flux. These results suggest cautious interpretation of mutant Ca(2+)/H(+) exchanger phenotypes that may be due to either perturbed Ca(2+) or H(+) transport.

  19. /sup 1/H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla

    Energy Technology Data Exchange (ETDEWEB)

    MacD. Stewart, J.M.; Grisham, C.M.

    1988-06-28

    /sup 1/H nuclear magnetic relaxation measurements have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH/sub 3/)/sub 4/ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH/sub 4/)/sub 4/ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase and that Mn/sup 2 +/ bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na-K-ATPase. From the paramagnetic effect of Mn/sup 2 +/ bound to the APTase on the longitudinal relaxation rates of the protons of Co(NH/sub 3/)/sub 4/ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous /sup 31/P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35/sup 0/, and the conformation of the ribose ring is slightly N-type. The bound Mn/sup 2 +/ lies above and in the plane of the adenine ring. The distances from Mn/sup 2 +/ to N/sub 6/ and N/sub 7/ are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.

  20. Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Rigos, C.F.; Tedesco, A.C.; Ciancaglini, P. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Filosofia, Ciencias e Letras. Dept. de Quimica; Santos, H.L. [Universidade Federal de Sao Joao Del Rei (UFSJ), MG (Brazil)

    2008-07-01

    Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.

  1. Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

    International Nuclear Information System (INIS)

    Rigos, C.F.; Tedesco, A.C.; Ciancaglini, P.

    2008-01-01

    Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes

  2. Isolation and characterization of DNA-dependent ATPases from the Novikoff Hepatoma

    International Nuclear Information System (INIS)

    Thomas, D.C.

    1984-01-01

    Four DNA-dependent ATPases have been purified to apparent homogeneity from extracts of the Novikoff Hepatoma, and named ATPases II, III, IV, and V. The physical and enzymological properties of ATPases II, III, and V are nearly identical, and from tryptic peptide mapping these proteins were determined to be related, though they are still chromatographically distinct; all appear to be dimers. ATPaseIV is unique among the ATPases, and is probably a monomer. ATPase V appears much more stable to thermal inactivation than the similar curves generated by ATPases II, and III. ATPase IV, however, projects of a heat-inactivation curve intermediate to these two types. ATPase II is labelled to a much higher degree than the others when treated with a heterologous protein kinase using gamma-[ 32 P]-ATP. When ATPase II was treated with this kinase, and subsequently run over a DNA-cellulose column, the profile of ATPase II was found to contain small peaks of activity in the positions where ATPases III and V normally elute, suggesting that ATPase II may be a dephosphorylated form of the other two. The ATPases have been extensively characterized with respect to reaction products and requirements, substrate utilization, DNA effector requirements, and effects of ATP analogs

  3. Proton permeation of lipid bilayers.

    Science.gov (United States)

    Deamer, D W

    1987-10-01

    Proton permeation of the lipid bilayer barrier has two unique features. First, permeability coefficients measured at neutral pH ranges are six to seven orders of magnitude greater than expected from knowledge of other monovalent cations. Second, proton conductance across planar lipid bilayers varies at most by a factor of 10 when pH is varied from near 1 to near 11. Two mechanisms have been proposed to account for this anomalous behavior: proton conductance related to contaminants of lipid bilayers, and proton translocation along transient hydrogen-bonded chains (tHBC) of associated water molecules in the membrane. The weight of evidence suggests that trace contaminants may contribute to proton conductance across planar lipid membranes at certain pH ranges, but cannot account for the anomalous proton flux in liposome systems. Two new results will be reported here which were designed to test the tHBC model. These include measurements of relative proton/potassium permeability in the gramicidin channel, and plots of proton flux against the magnitude of pH gradients. (1) The relative permeabilities of protons and potassium through the gramicidin channel, which contains a single strand of hydrogen-bonded water molecules, were found to differ by at least four orders of magnitude when measured at neutral pH ranges. This result demonstrates that a hydrogen-bonded chain of water molecules can provide substantial discrimination between protons and other cations. It was also possible to calculate that if approximately 7% of bilayer water was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. (2) The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result that is consistent with one of three alternative tHBC models for proton conductance described by Nagle elsewhere in this volume.

  4. Recombinase, chromosomal translocations and lymphoid neoplasia: targeting mistakes and repair failures.

    Science.gov (United States)

    Marculescu, Rodrig; Vanura, Katrina; Montpellier, Bertrand; Roulland, Sandrine; Le, Trang; Navarro, Jean-Marc; Jäger, Ulrich; McBlane, Fraser; Nadel, Bertrand

    2006-09-08

    A large number of lymphoid malignancies is characterized by specific chromosomal translocations, which are closely linked to the initial steps of pathogenesis. The hallmark of these translocations is the ectopic activation of a silent proto-oncogene through its relocation at the vicinity of an active regulatory element. Due to the unique feature of lymphoid cells to somatically rearrange and mutate receptor genes, and to the corresponding strong activity of the immune enhancers/promoters at that stage of cell development, B- and T-cell differentiation pathways represent propitious targets for chromosomal translocations and oncogene activation. Recent progress in the understanding of the V(D)J recombination process has allowed a more accurate definition of the translocation mechanisms involved, and has revealed that V(D)J-mediated translocations result both from targeting mistakes of the recombinase, and from illegitimate repair of the V(D)J recombination intermediates. Surprisingly, V(D)J-mediated translocations turn out to be restricted to two specific sub-types of lymphoid malignancies, T-cell acute lymphoblastic leukemias, and a restricted set of mature B-cell Non-Hodgkin's lymphomas.

  5. Effects of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in Wistar rats.

    Science.gov (United States)

    Olatunji, Lawrence A; Usman, Taofeek O; Adebayo, Joseph O; Olatunji, Victoria A

    2012-09-01

    To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in rats. The 25 and 50 mg/(kg·d) of aqueous extracts of H. sabdariffa were respectively given to rats in the experimental groups for 28 d, and rats in the control group received an appropriate volume of distilled water as vehicle. Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in the kidney were assayed by spectrophotometric method. Administrations of 25 and 50 mg/(kg·d) of aqueous extract of H. sabdariffa significantly decreased the Ca(2+)-Mg(2+)-ATPase activity in the kidney of rats (Psabdariffa may preserve the renal function despite a decreased renal Ca(2+)-Mg(2+)-ATPase activity.

  6. Enhanced target normal sheath acceleration of protons from intense laser interaction with a cone-tube target

    Directory of Open Access Journals (Sweden)

    K. D. Xiao

    2016-01-01

    Full Text Available Laser driven proton acceleration is proposed to be greatly enhanced by using a cone-tube target, which can be easily manufactured by current 3D-print technology. It is observed that energetic electron bunches are generated along the tube and accelerated to a much higher temperature by the combination of ponderomotive force and longitudinal electric field which is induced by the optical confinement of the laser field. As a result, a localized and enhanced sheath field is produced at the rear of the target and the maximum proton energy is about three-fold increased based on the two-dimentional particle-in-cell simulation results. It is demonstrated that by employing this advanced target scheme, the scaling of the proton energy versus the laser intensity is much beyond the normal target normal sheath acceleration (TNSA case.

  7. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus Emanuel; Liu, Xiang-Yu; Skjørringe, Tina

    2011-01-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, ......Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu...

  8. Goatpoxvirus ATPase activity is increased by dsDNA and decreased by zinc ion.

    Science.gov (United States)

    Lee, Ming-Liang; Hsu, Wei-Li; Wang, Chi-Young; Chen, Hui-Yu; Lin, Fong-Yuan; Chang, Ming-Huang; Chang, Hong-You; Wong, Min-Liang; Chan, Kun-Wei

    2016-10-01

    Viral-encoded ATPase can act as a part of molecular motor in genome packaging of DNA viruses, such as vaccinia virus and adenovirus, by ATP hydrolysis and interaction with DNA. Poxviral ATPase (also called A32) is involved in genomic double-stranded DNA (dsDNA) encapsidation, and inhibition of the expression of A32 causes formation of immature virions lacking viral DNA. However, the role of A32 in goatpoxvirus genome packaging and its dsDNA binding property are not known. In this study, purified recombinant goatpoxvirus A32 protein (rA32) was examined for its dsDNA binding property as well as the effect of dsDNA on ATP hydrolysis. We found that rA32 could bind dsDNA, and its ATPase activity was significant increased with dsDNA binding. Effects of magnesium and calcium ions on ATP hydrolysis were investigated also. The ATPase activity was dramatically enhanced by dsDNA in the presence of Mg(2+); in contrast, ATPase function was not altered by Ca(2+). Furthermore, the enzyme activity of rA32 was completely blocked by Zn(2+). Regarding DNA-protein interaction, the rA32-ATP-Mg(2+) showed lower dsDNA binding affinity than that of rA32-ATP-Ca(2+). The DNA-protein binding was stronger in the presence of zinc ion. Our results implied that A32 may play a role in viral genome encapsidation and DNA condensation.

  9. The gastric H,K-ATPase blocker lansoprazole is an inhibitor of chloride channels

    Science.gov (United States)

    Schmarda, Andreas; Dinkhauser, Patrick; Gschwentner, Martin; Ritter, Markus; Fürst, Johannes; Scandella, Elke; Wöll, Ewald; Laich, Andreas; Rossmann, Heidi; Seidler, Ursula; Lang, Florian; Paulmichl, Markus

    2000-01-01

    It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances – which are known to block the H,K-ATPase – could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels – characterized in many different cell types – show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells. PMID:10711360

  10. The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

    OpenAIRE

    Ishii, T; Takeyasu, K

    1993-01-01

    Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity,...

  11. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2010-11-19

    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  12. Tomato leaf curl Kerala virus (ToLCKeV AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1

    Directory of Open Access Journals (Sweden)

    Mukherjee Sunil K

    2010-06-01

    Full Text Available Abstract Background Geminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study. Results AC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro. Conclusions Highly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.

  13. Enhancement of proton transfer in ion channels by membrane phosphate headgroups.

    Science.gov (United States)

    Wyatt, Debra L; de Godoy, Carlos Marcelo G; Cukierman, Samuel

    2009-05-14

    The transfer of protons (H+) in gramicidin (gA) channels is markedly distinct in monoglyceride and phospholipid membranes. In this study, the molecular groups that account for those differences were investigated using a new methodology. The rates of H+ transfer were measured in single gA channels reconstituted in membranes made of plain ceramides or sphingomyelins and compared to those in monoglyceride and phospholipid bilayers. Single-channel conductances to protons (gH) were significantly larger in sphingomyelin than in ceramide membranes. A novel and unsuspected finding was that H+ transfer was heavily attenuated or completely blocked in ceramide (but not in sphingomyelin) membranes in low-ionic-strength solutions. It is reasoned that H-bond dynamics at low ionic strengths between membrane ceramides and gA makes channels dysfunctional. The rate of H+ transfer in gA channels in ceramide membranes is significantly higher than that in monoglyceride bilayers. This suggests that solvation of the hydrophobic surface of gA channels by two acyl chains in ceramides stabilizes the gA channels and the water wire inside the pore, leading to an enhancement of H+ transfer in relation to that occurring in monoglyceride membranes. gH values in gA channels are similar in ceramide and monoglyceride bilayers and in sphingomyelin and phospholipid membranes. It is concluded that phospho headgroups in membranes have significant effects on the rate of H+ transfer at the membrane gA channel/solution interfaces, enhancing the entry and exit rates of protons in channels.

  14. Probiotic Mixture Golden Bifido Prevents Neonatal Escherichia coli K1 Translocation via Enhancing Intestinal Defense

    Directory of Open Access Journals (Sweden)

    Qing Zeng

    2017-09-01

    Full Text Available Escherichia coli (E. coli K1 sepsis and meningitis is a severe infection characterized by high mortality in neonates. Successful colonization and translocation across the intestinal mucosa have been regarded as the critical steps for E. coli K1 sepsis and meningitis. We recently reported that the probiotic mixture, Golden Bifido (containing live Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus, LBS has a preventive role against neonatal E. coli K1 bacteremia and meningitis. However, the interaction between the neonatal gut barrier, probiotics and E. coli K1 is still not elucidated. The present study aims to investigate how LBS exerts its protective effects on neonatal gut barrier during E. coli K1 infection. The beneficial effects of LBS were explored in vitro and in vivo using human colon carcinoma cell lines HT-29 and rat model of neonatal E. coli K1 infection, respectively. Our results showed that stimulation with E. coli K1 was able to cause intestinal barrier dysfunction, which were reflected by E. coli K1-induced intestinal damage and apoptosis of intestinal epithelial cells, reduction of mucin, immunoglobulin A (IgA and tight junction proteins expression, as well as increase in intestinal permeability, all these changes facilitate E. coli K1 intestinal translocation. However, these changes were alleviated when HT-29 cells were treated with LBS before E. coli K1 infection. Furthermore, we found that LBS-treated neonatal rats (without E. coli K1 infection have showed higher production of mucin, ZO-1, IgA, Ki67 in intestinal mucosa as well as lower intestinal permeability than that of non-treated rats, indicating that LBS could accelerate the development of neonatal intestinal defense. Taken together, our results suggest that enhancement of the neonatal intestinal defense to fight against E. coli K1 translocation could be the potential mechanism to elucidate how LBS confers a protective effect against neonatal E

  15. Uptake, translocation, and debromination of polybrominated diphenyl ethers in maize

    Institute of Scientific and Technical Information of China (English)

    Moming Zhao; Shuzhen Zhang; Sen Wang; Honglin Huang

    2012-01-01

    Uptake,translocation and debromination of three polybrominated diphenyl ethers(PBDEs),BDE-28,-47 and-99,in maize were studied in a hydroponic experiment.Roots took up most of the PBDEs in the culture solutions and more highly brominated PBDEs had a stronger uptake capability.PBDEs were detected in the stems and leaves of maize after exposure but rarely detected in the blank control plants.Furthermore,PBDE concentrations decreased from roots to stems and then to leaves,and a very clear decreasing gradient was found in segments upwards along the stem.These altogether provide substantiating evidence for the acropetal translocation of PBDEs in maize.More highly brominated PBDEs were translocated with more difficulty.Radial translocation of PBDEs from nodes to sheath inside maize was also observed.Both acropetal and radial translocations were enhanced at higher transpiration rates,suggesting that PBDE transport was probably driven by the transpiration stream.Debromination of PBDEs occurred in all parts of the maize,and debromination patterns of different parent PBDEs and in different parts of a plant were similar but with some differences.This study for the first time provides direct evidence for the acropetal translocation of PBDEs within plants,elucidates the process of PBDE transport and clarifies the debromination products of PBDEs in maize.

  16. Reversible pH-dependent activation/inactivation of CF(1-ATPase of spinach chloroplasts

    Directory of Open Access Journals (Sweden)

    A. P. Khomochkin

    2017-08-01

    Full Text Available The aim of the work was to study the reverse pH-dependent regulation of the enzymatic activity of the catalytic part of ATP synthase (EC 3.6.3.14 of chloroplast – coupling factor CF1. It was shown that the short-term incubation of isolated CF1 in the media with pH 4.5 or 3.5 leads to inactivation of Ca2+-ATPase, which is rapidly (t1/2 ~ 1 min restored in the medium containing 0.5-10 mM bicarbonate at pH 7.8. After acid treatment, the rate of Mg2+-ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С. The increase in Ca2+– and Mg2+-АТР activity of CF1 associated with the addition of NaHCO3 solution was completely eliminated after the introduction of 50 mM acetazolamide – a specific inhibitor of carbonic anhydrase. The obtained results suggest the existence of the bound bicarbonate in the CF1 structure, which apparently participates in proton transfer.

  17. Multiple aspects of ATP-dependent nucleosome translocation by RSC and Mi-2 are directed by the underlying DNA sequence.

    Directory of Open Access Journals (Sweden)

    Joke J F A van Vugt

    Full Text Available BACKGROUND: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. METHODOLOGY/PRINCIPAL FINDINGS: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type and native RSC from yeast (SWI/SNF-type repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. CONCLUSIONS/SIGNIFICANCE: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase

  18. Inactivation of mitochondrial ATPase by ultraviolet light

    International Nuclear Information System (INIS)

    Chavez, E.; Cuellar, A.

    1984-01-01

    The present work describes experiments that show that far-ultraviolet irradiation induce the inhibition of ATPase activity in both membrane-bound and soluble F1. It was also found that ultraviolet light promotes the release of tightly bound adenine nucleotides from F1-ATPase. Experiments carried out with submitochondrial particles indicate that succinate partially protects against these effects of ultraviolet light. Titration of sulfhydryl groups in both irradiated submitochondrial particles and soluble F1-ATPase indicates that a conformational change induced by photochemical modifications of amino acid residues appears involved in the inactivation of the enzyme. Finally, experiments are described which show that the tyrosine residue located in the active site of F1-ATPase is modified by ultraviolet irradiation

  19. Evidence of two different Na/sup +/-dependent (/sup 3/H)-ouabain binding sites of a Na/sup +/-K/sup +/-ATPase of guinea-pig hearts

    Energy Technology Data Exchange (ETDEWEB)

    Fricke, U; Klaus, W [Koeln Univ. (Germany, F.R.)

    1977-11-01

    The influence of various Na/sup +/ concentrations on (/sup 3/H)-ouabain binding was studied in experiments on a microsomal Na/sup +/-K/sup +/-adenosine triphosphatase (ATPase) from guinea-pig hearts. The ATP-independent cardiac glycoside binding was not influenced by increasing Na/sup +/ concentrations. However, a good correlation was found between the ATP-dependent (/sup 3/H)-ouabain binding and Na/sup +/ concentration. A more detailed analysis of these results revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na/sup +/ concentrations, (Ksub(0.5) = 4.5 mM); this type of (/sup 3/H)-ouabain binding was strongly correlated to the Na/sup +/ concentration necessary for half maximum phosphorylation (Ksub(0.5) = 1 mM). The other ouabain binding process was predominant at high Na/sup +/ concentrations (Ksub(0.5 = 69 mM). On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na/sup +/-K/sup +/-ATPase is proposed, assuming two different binding sites for cardiac glycosides (E/sub 2/ -P and Esub(l) -P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.

  20. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  1. Kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.L.; van Kooten, O.; Vredenberg, W.J.

    1985-08-01

    The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash-induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10(-8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.

  2. NCBI nr-aa BLAST: CBRC-AGAM-07-0045 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0045 ref|ZP_01051647.1| Heavy metal translocating P-type ATPase [Tenac...ibaculum sp. MED152] gb|EAQ41075.1| Heavy metal translocating P-type ATPase [Polaribacter dokdonensis MED152] ZP_01051647.1 1e-132 63% ...

  3. Sequence Classification: 501435 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH TMB TMB TMB Non-TMB >gi|66048305|ref|YP_238146.1| Heavy metal-(Cd/Co/Hg.../Pb/Zn)-translocating P-type ATPase:Heavy metal translocating P-type ATPase || http://www.ncbi.nlm.nih.gov/protein/66048305 ...

  4. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    International Nuclear Information System (INIS)

    Nakajima, Hideaki; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-01-01

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix

  5. Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation*

    Science.gov (United States)

    Burgess, Jamie L.; Burgess, R. Alan; Morales, Yalemi; Bouvang, Jenna M.; Johnson, Sean J.; Dickenson, Nicholas E.

    2016-01-01

    Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. PMID:27770024

  6. Proton energy spectra during ground level enhancements as measured by EPHIN aboard SOHO

    Energy Technology Data Exchange (ETDEWEB)

    Heber, Bernd; Kuehl, Patrick; Klassen, Andreas; Dresing, Nina [Christian-Albrechts-Universitaet zu Kiel, 24118 Kiel (Germany); Gomez-Herrero, Raul [Universidad de Alcala (Spain)

    2016-07-01

    Ground Level Enhancements (GLEs) are solar energetic particle (SEP) events that are recorded by ground-based instrumentation. The energy of the particles is so high that they produce secondary particles in the Earth's atmosphere, i.e. protons and neutrons, which are detected as sudden increases in cosmic ray intensities measured by e.g. neutron monitors. Since the launch of SOHO in December 1995 the neutron monitor network recorded 16 GLEs. The Electron Proton Helium INstrument on board SOHO has been designed to measure protons and helium up to 53 MeV/nucleon as well as electrons up to 8.3 MeV. Above these energies, particles penetrate all detector elements and thus, a separation between different particle species becomes more complicated. Recently we developed a method that allows deriving the energy spectrum for penetrating protons up to more than 1 GeV. In this contribution we present the proton energy spectra and time profiles of above mentioned GLEs and compare them to previous measurements. Although there are differences of up to a factor two the overall shape of the energy spectra agree surprisingly well. Thus it has been demonstrated that EPHIN measurements are a valuable tool for understanding GLE.

  7. The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP*

    Science.gov (United States)

    Niwa, Hajime; Ewens, Caroline A.; Tsang, Chun; Yeung, Heidi O.; Zhang, Xiaodong; Freemont, Paul S.

    2012-01-01

    p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97A232E, having three times higher activity. Further mutagenesis of p97A232E shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97A232E suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive. PMID:22270372

  8. The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP.

    Science.gov (United States)

    Niwa, Hajime; Ewens, Caroline A; Tsang, Chun; Yeung, Heidi O; Zhang, Xiaodong; Freemont, Paul S

    2012-03-09

    p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97(A232E), having three times higher activity. Further mutagenesis of p97(A232E) shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97(A232E) suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.

  9. Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na/sup +/,K/sup +/-ATPase inhibitors in acutely volume-expanded hog

    Energy Technology Data Exchange (ETDEWEB)

    Tamura, M.; Harris, T.M.; Higashimori, K.; Sweetman, B.J.; Blair, I.A.; Inagami, T.

    1987-05-19

    Na/sup +/,K/sup +/-ATPase inhibitors activities against the specific binding of ouabain to Na/sup +/,K/sup +/-ATPase and /sup 86/Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as ..gamma..-arachidoyl- (LPCA(..gamma..), 34%), ..beta..-arachidoyl- (LPCA(..beta..), 4%), ..gamma..-linoleoyl- (LPCL, 33%), and ..gamma..-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of ..gamma..-docosapentaenoyl-, ..gamma..-eicosatrienoyl-, and ..gamma..palmitoyllysophosphatidylcholine were also detected by both FAB mass and /sup 1/H NMR spectrometric studies. The inhibition of Na/sup +/,K/sup +/-ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and /sup 86/Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest that ..gamma..-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na/sup +/,K/sup +/-ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme.

  10. NMR spectroscopic studies of membrane-bound biological systems

    International Nuclear Information System (INIS)

    Hohlweg, W.

    2013-01-01

    In the course of this thesis, biological NMR spectroscopy was employed in studying membrane-bound peptides and proteins, for which structural information is still comparatively hard to obtain. Initial work focused on various model peptides bound to membrane-mimicking micelles, studying the protonation state of arginine in a membrane environment. Strong evidence for a cation-π complex was found in TM7, a peptide which forms the seventh transmembrane helix of subunit a of the vacuolar-type H+-ATPase (V-ATPase). V-ATPase is a physiologically highly relevant proton pump, which is present in intracellular membranes of all eukaryotic organisms, as well as the plasma membrane of several specialized cells. Loss of functional V-ATPase is associated with human diseases such as osteopetrosis, distal renal tubular acidosis or the spreading of cancer. V-ATPase is considered a potential drug target in the treatment of osteoporosis and cancer, or in the development of novel contraceptives. Results from NMR solution structure determination, NMR titration experiments, paramagnetic relaxation enhancement experiments and tryptophan fluorescence spectroscopy confirm the existence of a buried cation-? complex formed between arginine residue R735, which is essential for proton transport, and neighbouring tryptophan and tyrosine residues. In vivo experiments in the yeast Saccharomyces cerevisiae using selective growth tests and fluorescence microscopy showed that formation of the cation-π complex is essential for V-ATPase function. Deletion of both aromatic residues, as well as only the one tryptophan residue leads to growth defects and inability to maintain vacuolar pH homeostasis. These findings shine new light on the still elusive mechanism of proton transport in V-ATPase, and show that arginine R735 may be directly involved in proton transfer across the membrane. (author) [de

  11. Regulation of cardiac remodeling by cardiac Na/K-ATPase isoforms

    Directory of Open Access Journals (Sweden)

    Lijun Catherine Liu

    2016-09-01

    Full Text Available Cardiac remodeling occurs after cardiac pressure/volume overload or myocardial injury during the development of heart failure and is a determinant of heart failure. Preventing or reversing remodeling is a goal of heart failure therapy. Human cardiomyocyte Na+/K+-ATPase has multiple α isoforms (1-3. The expression of the α subunit of the Na+/K+-ATPase is often altered in hypertrophic and failing hearts. The mechanisms are unclear. There are limited data from human cardiomyocytes. Abundant evidences from rodents show that Na+/K+-ATPase regulates cardiac contractility, cell signaling, hypertrophy and fibrosis. The α1 isoform of the Na+/K+-ATPase is the ubiquitous isoform and possesses both pumping and signaling functions. The α2 isoform of the Na+/K+-ATPase regulates intracellular Ca2+ signaling, contractility and pathological hypertrophy. The α3 isoform of the Na+/K+-ATPase may also be a target for cardiac hypertrophy. Restoration of cardiac Na+/K+-ATPase expression may be an effective approach for prevention of cardiac remodeling. In this article, we will overview: (1 the distribution and function of isoform specific Na+/K+-ATPase in the cardiomyocytes. (2 the role of cardiac Na+/K+-ATPase in the regulation of cell signaling, contractility, cardiac hypertrophy and fibrosis in vitro and in vivo. Selective targeting of cardiac Na+/K+-ATPase isoform may offer a new target for the prevention of cardiac remodeling.

  12. Proton channels and exchangers in cancer.

    Science.gov (United States)

    Spugnini, Enrico Pierluigi; Sonveaux, Pierre; Stock, Christian; Perez-Sayans, Mario; De Milito, Angelo; Avnet, Sofia; Garcìa, Abel Garcìa; Harguindey, Salvador; Fais, Stefano

    2015-10-01

    Although cancer is characterized by an intratumoral genetic heterogeneity, a totally deranged pH control is a common feature of most cancer histotypes. Major determinants of aberrant pH gradient in cancer are proton exchangers and transporters, including V-ATPase, Na+/H+ exchanger (NHE), monocarboxylate transporters (MCTs) and carbonic anhydrases (CAs). Thanks to the activity of these proton transporters and exchangers, cancer becomes isolated and/or protected not only from the body reaction against the growing tumor, but also from the vast majority of drugs that when protonated into the acidic tumor microenvironment do not enter into cancer cells. Proton transporters and exchangers represent a key feature tumor cells use to survive in the very hostile microenvironmental conditions that they create and maintain. Detoxifying mechanisms may thus represent both a key survival option and a selection outcome for cells that behave as unicellular microorganisms rather than belonging to an organ, compartment or body. It is, in fact, typical of malignant tumors that, after a clinically measurable yet transient initial response to a therapy, resistant tumor clones emerge and proliferate, thus bursting a more malignant behavior and rapid tumor progression. This review critically presents the background of a novel and efficient approach that aims to fight cancer through blocking or inhibiting well characterized proton exchangers and transporters active in human cancer cells. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. H+-ATPase activity from storage tissue of Beta vulgaris. IV. N,N'-dicyclohexylcarbodiimide binding and inhibition of the plasma membrane H+-ATPase

    International Nuclear Information System (INIS)

    Oleski, N.A.; Bennett, A.B.

    1987-01-01

    The molecular weight and isoelectric point of the plasma membrane H + -ATPase from red beet storage tissue were determined using N,N'-dicyclohexylcarbodiimide (DCCD) and a H + -ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [ 14 C]-DCCD at 0 0 C, a single 97,000 dalton protein was visualized on a fluorography of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [ 14 C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H + -ATPase. An antibody raised against the plasma membrane H + -ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H + -ATPase to be 6.5

  14. Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-ichiro; Takahashi, Koji; Ishizaki, Kimitsune; Kohchi, Takayuki; Kinoshita, Toshinori

    2012-06-01

    The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.

  15. Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na(+) and K(+) across the Mycobacterium smegmatis plasma membrane.

    Science.gov (United States)

    Ayala-Torres, Carlos; Novoa-Aponte, Lorena; Soto, Carlos Y

    2015-07-01

    Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme

    International Nuclear Information System (INIS)

    Kasho, V.N.; Boyer, P.D.

    1989-01-01

    Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F 1 , F 0 -ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F 1 ,F 0 -ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P i as medium ATP concentration was lowered was determined by 18 O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P i formed as ATP concentration is decreased to the micromolar range. The F 1 ,F 0 -ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F 1 -type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F 1 ,F 0 -ATPases

  17. O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase

    International Nuclear Information System (INIS)

    Kim, M.S.; Akera, T.

    1987-01-01

    The role of O2 free radicals in the reduction of sarcolemmal Na+-K+-ATPase, which occurs during reperfusion of ischemic heart, was examined in isolated guinea pig heart using exogenous scavengers of O2 radicals and an inhibitor of xanthine oxidase. Ischemia and reperfusion reduced Na+-K+-ATPase activity and specific [3H]ouabain binding to the enzyme in ventricular muscle homogenates and also markedly lowered sodium pump activity estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices. These effects of ischemia and reperfusion were prevented to various degrees by O2-radical scavengers, such as superoxide dismutase, catalase, dimethyl-sulfoxide, histidine, or vitamin E or by the xanthine oxidase inhibitor, allopurinol. The degree of protection afforded by these agents paralleled that of reduction in enhanced lipid peroxidation of myocardial tissue as estimated from malondialdehyde production. These results strongly suggest that O2 radicals play a crucial role in the injury to sarcolemmal Na+-K+-ATPase during reperfusion of ischemic heart

  18. O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Kim, M.S.; Akera, T.

    1987-02-01

    The role of O2 free radicals in the reduction of sarcolemmal Na+-K+-ATPase, which occurs during reperfusion of ischemic heart, was examined in isolated guinea pig heart using exogenous scavengers of O2 radicals and an inhibitor of xanthine oxidase. Ischemia and reperfusion reduced Na+-K+-ATPase activity and specific (3H)ouabain binding to the enzyme in ventricular muscle homogenates and also markedly lowered sodium pump activity estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices. These effects of ischemia and reperfusion were prevented to various degrees by O2-radical scavengers, such as superoxide dismutase, catalase, dimethyl-sulfoxide, histidine, or vitamin E or by the xanthine oxidase inhibitor, allopurinol. The degree of protection afforded by these agents paralleled that of reduction in enhanced lipid peroxidation of myocardial tissue as estimated from malondialdehyde production. These results strongly suggest that O2 radicals play a crucial role in the injury to sarcolemmal Na+-K+-ATPase during reperfusion of ischemic heart.

  19. Biological mechanisms and translocation kinetics of particulate plutonium

    International Nuclear Information System (INIS)

    Bruenger, F.W.; Stevens, W.; Atherton, D.R.; Roswell, R.L.; Smith, J.M.

    1981-01-01

    The dissolution and elimination of particulate 239 Pu from its initial sites of deposition in phagocytic organs (the liver, spleen, and lung), as well as its translocation and redeposition in soft tissue organs and skeleton have been investigated. Beagles were injected intravenously with particulate Pu and sacrificed sequentially at times ranging from 33 to 830 days after injection. Equations that describe the overall retention of Pu in liver, spleen, lung, and bone were calculated. Plutonium mobilized from these organs either re-entered the blood stream and redeposited in the skeleton and liver parenchyma or was excreted. The protracted translocation of Pu to bone surfaces potentially exposes all cells involved in osteogenesis to continuous α-radiation, a situation that could enhance the hazard of developing osteosarcoma. A kinetic model that describes the translocation of Pu from the phagocytic compartments to blood and its subsequent redistribution to bone, liver, and other organs was formulated

  20. Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

    Science.gov (United States)

    Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

    2008-11-01

    We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

  1. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

    Science.gov (United States)

    Banerjee, Subhrajit; Kane, Patricia M

    2017-09-15

    Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H + -ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P 2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of V o a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. Conditions of activation of yeast plasma membrane ATPase.

    Science.gov (United States)

    Sychrová, H; Kotyk, A

    1985-04-08

    The in vivo activation of the H+-ATPase of baker's yeast plasma membrane found by Serrano in 1983 was demonstrated with D-glucose aerobically and anaerobically (as well as in a respiration-deficient mutant) and, after suitable induction, with maltose, trehalose, and galactose. The activated but not the control ATPase was sensitive to oligomycin. No activation was possible in a cell-free extract with added glucose. The ATPase was not activated in yeast protoplasts which may account for the absence of glucose-stimulated secondary active transports in these wall-less cells and provide support for a microscopic coupling between ATPase activity and these transports in yeast cells.

  3. Rethinking the Combination of Proton Exchanger Inhibitors in Cancer Therapy.

    Science.gov (United States)

    Iessi, Elisabetta; Logozzi, Mariantonia; Mizzoni, Davide; Di Raimo, Rossella; Supuran, Claudiu T; Fais, Stefano

    2017-12-23

    Microenvironmental acidity is becoming a key target for the new age of cancer treatment. In fact, while cancer is characterized by genetic heterogeneity, extracellular acidity is a common phenotype of almost all cancers. To survive and proliferate under acidic conditions, tumor cells up-regulate proton exchangers and transporters (mainly V-ATPase, Na⁺/H⁺ exchanger (NHE), monocarboxylate transporters (MCTs), and carbonic anhydrases (CAs)), that actively extrude excess protons, avoiding intracellular accumulation of toxic molecules, thus becoming a sort of survival option with many similarities compared with unicellular microorganisms. These systems are also involved in the unresponsiveness or resistance to chemotherapy, leading to the protection of cancer cells from the vast majority of drugs, that when protonated in the acidic tumor microenvironment, do not enter into cancer cells. Indeed, as usually occurs in the progression versus malignancy, resistant tumor clones emerge and proliferate, following a transient initial response to a therapy, thus giving rise to more malignant behavior and rapid tumor progression. Recent studies are supporting the use of a cocktail of proton exchanger inhibitors as a new strategy against cancer.

  4. Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

    Science.gov (United States)

    Holm, Rikke; Khandelwal, Jaanki; Einholm, Anja P; Andersen, Jens P; Artigas, Pablo; Vilsen, Bente

    2017-01-10

    Na + ,K + -ATPase and H + ,K + -ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H + ,K + -ATPase avoids binding of Na + at the site corresponding to the Na + -specific site of the Na + ,K + -ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na + ,K + -ATPase with arginine, present in the H + ,K + -ATPase at the corresponding position, converted the normal 3Na + :2K + :1ATP stoichiometry of the Na + ,K + -ATPase to electroneutral 2Na + :2K + :1ATP stoichiometry similar to the electroneutral transport mode of the H + ,K + -ATPase. The electroneutral C932R mutant of the Na + ,K + -ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K + uptake. Only a relatively minor reduction of apparent Na + affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na + affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine + group of the M8 arginine replaces Na + at the third site, thus preventing Na + binding there, although allowing Na + to bind at the two other sites and become transported. Hence, in the H + ,K + -ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

  5. Proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley-powdery mildew interaction

    DEFF Research Database (Denmark)

    Zhou, F.S.; Andersen, C.H.; Burhenne, K.

    2000-01-01

    We propose a model for activation of the epidermal cell hypersensitive response (HR) in the barley/powdery mildew interaction. The model suggests that the plasma membrane proton pump (H+-ATPase) of epidermal cells is activated following penetration by an avirulent powdery mildew fungus...... in the incompatible interaction; (4) race-specific proton extrusion is observed underneath epidermal tissue detached from leaves inoculated 15 h earlier; and (5) treatment of leaves with fusicoccin, an activator of the plasma membrane H+-ATPase, increases the number of HR-cells in the compatible interaction........ This will cause an acidification of the apoplast towards the mesophyll cells, thereby activating generation of H2O2 from the mesophyll, which subsequently triggers the epidermal cell to undergo HR. The model is supported by the following data: (1) the earliest HR-related H2O2 is found in the attachment zones...

  6. Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function.

    Science.gov (United States)

    Kondabagil, Kiran; Dai, Li; Vafabakhsh, Reza; Ha, Taekjip; Draper, Bonnie; Rao, Venigalla B

    2014-11-01

    The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH(-) nucleophile. The ATPase Walker B motif, thus, may be redefined as "β-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid". Copyright © 2014 Elsevier Inc. All rights reserved.

  7. The mechanism of Torsin ATPase activation.

    Science.gov (United States)

    Brown, Rebecca S H; Zhao, Chenguang; Chase, Anna R; Wang, Jimin; Schlieker, Christian

    2014-11-11

    Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.

  8. Sequence Classification: 119807 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|52081840|ref|YP_080631.1| Heavy met...al-(Cd/Co/Hg/Pb/Zn)-translocating P-type ATPase,Heavy metal translocating P-type ATPase || http://www.ncbi.nlm.nih.gov/protein/52081840 ...

  9. Sequence Classification: 501191 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|66048061|ref|YP_237902.1| Heavy met...al-(Cd/Co/Hg/Pb/Zn)-translocating P-type ATPase:Heavy metal translocating P-type ATPase || http://www.ncbi.nlm.nih.gov/protein/66048061 ...

  10. Modulation and Functional Role of the Orientations of the N- and P-Domains of Cu+ -Transporting ATPase along the Ion Transport Cycle.

    Science.gov (United States)

    Meng, Dan; Bruschweiler-Li, Lei; Zhang, Fengli; Brüschweiler, Rafael

    2015-08-18

    Ion transport of different P-type ATPases is regulated similarly through the interplay of multiple protein domains. In the presence of ATP, binding of a cation to the ion binding site in the transmembrane helices leads to the phosphorylation of the P-domain, allowing ion transfer across the membrane. The details of the mechanism, however, are not clear. Here, we report the modulation of the orientation between the N- and P-domains of Cu(+)-transporting ATPase along the ion transport cycle using high-resolution nuclear magnetic resonance spectroscopy in solution. On the basis of residual dipolar coupling measurements, it is found that the interdomain orientation (relative openness) of the N- and P-domains is distinctly modulated depending on the specific state of the N- and P-domains along the ion translocation cycle. The two domains' relative position in the apo state is semiopen, whereas it becomes closed upon binding of ATP to the N-domain. After phosphorylation of the P-domain and the release of ADP, the opening, however, becomes the widest among all the states. We reason such wide opening resulting from the departure of ADP prepares the N- and P-domains to accommodate the A-domain for interaction and, hence, promote ion transport and allow dephosphorylation of the P-domain. Such wide interdomain opening is abolished when an Asn to Asp mutation is introduced into the conserved DXXK motif located in the hinge region of the N- and P-domains of Cu(+)-ATPase, suggesting the indispensible role of the N- and P-interdomain orientation during ion transportation. Our results shed new light on the structural and mechanistic details of P-type ATPase function at large.

  11. Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses.

    Science.gov (United States)

    Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol; Auesukaree, Choowong

    2016-05-15

    During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic

  12. Translocation-based genetic sexing system to enhance the sterile insect technique against the melon fly (Diptera: Tephritidae)

    International Nuclear Information System (INIS)

    McCombs, S.D.; Lee, S.G.; Saul, S.H.

    1993-01-01

    The autosomal recessive bubble wing (bw) mutant was used to construct a translocation-based genetic sex sorting system in the melon fly, Bactrocera cucurbitae (Coquillett). The translocation stock has females with the bubble wing phenotype that are unable to fly, but the males are wild-type and fly normally. The bubble wing translocation strain has lower egg hatch, larval viability, and eclosion rates than the wild-type strain. Expression of the bubble wing trait is temperature-dependent, with high expression of the trait in 92% of adults at 23°C but in only 15% of adults at 28°C. This translocation-based sex sorting system is the only method available for automatic separation of male and female melon flies in sterile insect release programs

  13. Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin

    Directory of Open Access Journals (Sweden)

    Younes-Ibrahim M.

    1997-01-01

    Full Text Available On the basis of our report that a glycolipoprotein fraction (GLP extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1 GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2 Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3 GLP inhibits Na,K-ATPase from intact cells, and 4 GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 µg protein GLP/ml all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 µg protein GLP/ml for rat and rabbit, respectively, indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ± SEM; control: 23.8 ± 1.8; GLP, 88 µg protein/ml: 8.2 ± 0.9, demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis

  14. Myocardial Na,K-ATPase: Clinical aspects

    Science.gov (United States)

    Kjeldsen, Keld

    2003-01-01

    The specific binding of digitalis glycosides to Na,K-ATPase is used as a tool for Na,K-ATPase quantification with high accuracy and precision. In myocardial biopsies from patients with heart failure, total Na,K-ATPase concentration is decreased by around 40%; a correlation exists between a decrease in heart function and a decrease in Na,K-ATPase concentration. During digitalization, around 30% of remaining pumps are occupied by digoxin. Myocardial Na,K-ATPase is also influenced by other drugs used for the treatment of heart failure. Thus, potassium loss during diuretic therapy has been found to reduce myocardial Na,K-ATPase, whereas angiotensin-converting enzyme inhibitors may stimulate Na,K pump activity. Furthermore, hyperaldosteronism induced by heart failure has been found to decrease Na,K-ATPase activity. Accordingly, treatment with the aldosterone antagonist, spironolactone, may also influence Na,K-ATPase activity. The importance of Na,K pump modulation with heart disease, inhibition in digitalization and other effects of medication should be considered in the context of sodium, potassium and calcium regulation. It is recommended that digoxin be administered to heart failure patients who, after institution of mortality-reducing therapy, still have heart failure symptoms, and that the therapy be continued if symptoms are revealed or reduced. Digitalis glycosides are the only safe inotropic drugs for oral use that improve hemodynamics in heart failure. An important aspect of myocardial Na,K pump affection in heart disease is its influence on extracellular potassium (Ke) homeostasis. Two important aspects should be considered: potassium handling among myocytes, and effects of potassium entering the extracellular space of the heart via the bloodstream. It should be noted that both of these aspects of Ke homeostasis are affected by regulatory aspects, eg, regulation of the Na,K pump by physiological and pathophysiological conditions, as well as by medical

  15. Kidney in potassium depletion. I. Na+-K+-ATPase activity and [3H]ouabain binding in MCT

    International Nuclear Information System (INIS)

    Hayashi, M.; Katz, A.I.

    1987-01-01

    The effect of potassium depletion on renal Na + K + -ATPase was studied in rats. K depletion produced a striking, time-dependent increase in Na + -K + -ATPase activity of the outer medullary collecting tubules (inner stripe; MCT/sub is/). After 3 wk on the K-free diet, when the urine was almost potassium-free, Na + -K + -ATPase activity in MCT/sub is/ was over fourfold higher than in control animals. Repletion of potassium restored enzyme activity to base line within 7 days which corresponds to the catabolic rate of the renal enzyme, suggesting the cessation of enhanced synthesis that took place during K deprivation. Changes in Na + -K + -ATPase activity and aldosterone levels during both K depletion and repletion occurred in opposite directions and were therefore independent of each other. [ 3 H]Ouabain binding to intact MCT/sub is/, reflecting the number of pump sites on the basolateral membrane, was similar in K-depleted and control animals; in contrast, tubule permeabilization that exposes additional pump units to the ligand, unmasked a nearly fourfold increase in [ 3 H]ouabain binding in K-depleted rats, comparable to the increment in Na + -K + -ATPase activity. These results show that K depletion leads to a marked increase in Na + -K + -ATPase activity of MCT/sub is/, and suggest that the new enzyme units are located at a ouabain-inaccessible site in the intact tubule, i.e., either in an intracellular compartment or at the luminal membrane, where they may be involved in potassium reabsorption

  16. Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation.

    Science.gov (United States)

    Dixit, Aparna Banerjee; Ray, Krishanu; Black, Lindsay W

    2012-12-11

    Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

  17. The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase.

    NARCIS (Netherlands)

    Swarts, H.G.P.; Koenderink, J.B.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de

    2005-01-01

    We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a

  18. Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation.

    Science.gov (United States)

    Burgess, Jamie L; Burgess, R Alan; Morales, Yalemi; Bouvang, Jenna M; Johnson, Sean J; Dickenson, Nicholas E

    2016-12-09

    Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Regulation of the Plasma Membrane H+-ATPase

    DEFF Research Database (Denmark)

    Falhof, Janus

    The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

  20. Proton polarization in photo-excited aromatic molecule at room temperature enhanced by intense optical source and temperature control

    Energy Technology Data Exchange (ETDEWEB)

    Sakaguchi, S., E-mail: sakaguchi@phys.kyushu-u.ac.jp [Department of Physics, Kyushu University, Fukuoka 812-8581 (Japan); Uesaka, T. [RIKEN Nishina Center, Saitama 351-0198 (Japan); Kawahara, T. [Department of Physics, Toho University, Chiba 274-8510 (Japan); Ogawa, T. [RIKEN Advanced Science Institute, Saitama 351-0198 (Japan); Tang, L. [Center for Nuclear Study, University of Tokyo, Tokyo 113-0001 (Japan); Teranishi, T. [Department of Physics, Kyushu University, Fukuoka 812-8581 (Japan); Urata, Y.; Wada, S. [RIKEN Advanced Science Institute, Saitama 351-0198 (Japan); Wakui, T. [Cyclotron and Radioisotope Center (CYRIC), Tohoku University, Miyagi 980-8578 (Japan)

    2013-12-15

    Highlights: • Proton polarization in p-terphenyl at room-temperature is enhanced by a factor of 3. • Intense laser and temperature control are critically important for high polarization. • Optimization of time structure of laser pulse is effective for further improvement. -- Abstract: Proton polarization at room temperature, produced in a p-terphenyl crystal by using electron population difference in a photo-excited triplet state of pentacene, was enhanced by utilizing an intense laser with an average power of 1.5 W. It was shown that keeping the sample temperature below 300 K is critically important to prevent the rise of the spin–lattice relaxation rate caused by the laser heating. It is also reported that the magnitude of proton polarization strongly depends on the time structure of the laser pulse such as its width and the time interval between them.

  1. Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

    International Nuclear Information System (INIS)

    Knowles, A.F.; Lawrence, C.M.

    1986-01-01

    Plasma membranes of human oat cell carcinoma possess Mg 2+ - and Ca 2+ -dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca 2+ -ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca 2+ -ATPase activity is more easily inactivated by Triton X-100, while the Mg 2+ -ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca 2+ -ATPase and Mg 2+ -ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [ 3 H]FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca 2+ -ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg 2+ -ATPase has not been demonstrated, but is presently under investigation

  2. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls.

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T V; Alyethodi, Rafeeque R; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (P ATPase Β1, ATPase B2, and ATPase B3 is highly correlated (P ATPase beta family genes for cellular thermotolerance in cattle.

  3. Sperm Na+, K+-ATPase and Ca2+-ATPase activity: A preliminary study of comparison of swim up and density gradient centrifugation methods for sperm preparation

    Science.gov (United States)

    Lestari, Silvia W.; Larasati, Manggiasih D.; Asmarinah, Mansur, Indra G.

    2018-02-01

    As one of the treatment for infertility, the success rate of Intrauterine Insemination (IUI) is still relatively low. Several sperm preparation methods, swim-up (SU) and the density-gradient centrifugation (DGC) are frequently used to select for better sperm quality which also contribute to IUI failure. Sperm selection methods mainly separate the motile from the immotile sperm, eliminating the seminal plasma. The sperm motility involves the structure and function of sperm membrane in maintaining the balance of ion transport system which is regulated by the Na+, K+-ATPase, and Ca2+-ATPase enzymes. This study aims to re-evaluate the efficiency of these methods in selecting for sperm before being used for IUI and based the evaluation on sperm Na+,K+-ATPase and Ca2+-ATPase activities. Fourteen infertile men from couples who underwent IUI were involved in this study. The SU and DGC methods were used for the sperm preparation. Semen analysis was performed based on the reference value of World Health Organization (WHO) 2010. After isolating the membrane fraction of sperms, the Na+, K+-ATPase activity was defined as the difference in the released inorganic phosphate (Pi) with and without the existence of 10 mM ouabain in the reaction, while the Ca2+-ATPase was determined as the difference in Pi contents with and without the existence of 55 µm CaCl2. The prepared sperm demonstrated a higher percentage of motile sperm compared to sperm from the whole semen. Additionally, the percentage of motile sperm of post-DGC showed higher result than the sperm from post-SU. The velocity of sperm showed similar pattern with the percentage of motile sperm, in which the velocity of prepared sperm was higher than the sperm from whole semen. Furthermore, the sperm velocity of post-DGC was higher compared to the sperm from post-SU. The Na+, K+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Na+, K+-ATPase activity in the post DGC was higher than post SU. The Ca2

  4. Review: P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas

    DEFF Research Database (Denmark)

    Andersen, Jens P; Vestergaard, Anna L; Mikkelsen, Stine A

    2016-01-01

    group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca2+- and Na+/K+-ATPases. Hence, the P4-ATPase mechanism is quite similar...... on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms....

  5. Kinase-Mediated Regulation of P4-ATPases

    DEFF Research Database (Denmark)

    Frøsig, Merethe Mørch

    designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps...

  6. Thermophilic P-loop transport ATPases : Enzyme function and energetics at high temperature

    NARCIS (Netherlands)

    Pretz, Monika Gyöngyi

    2007-01-01

    Primary transport ATPases are divided into several superfamilies; amongst others including ATPases of the ABC transporter superfamily, the F-ATPase superfamily or the motor ATPases of the General Secretory (Sec) pathway. Motor proteins from these superfamilies show a low sequence similarity, except

  7. Functional size of vacuolar H+ pumps: Estimates from radiation inactivation studies

    International Nuclear Information System (INIS)

    Sarafian, V.; Poole, R.J.

    1991-01-01

    The PPase and the ATPase from red beet (Beta vulgaris) vacuolar membranes were subjected to radiation inactivation by a 60 Co source in both the native tonoplast and detergent-solubilized states, in order to determine their target molecular sizes. Analysis of the residual phosphohydrolytic and proton transport activities, after exposure to varying doses of radiation, yielded exponential relationships between the activities and radiation doses. The deduced target molecular sizes for PPase activity in native and solubilized membranes were 125kD and 259kD respectively and 327kD for H + -transport. This suggests that the minimum number of subunits of 67kD for PPi hydrolysis is two in the native state and four after Triton X-100 solubilization. At least four subunits would be required for H + -translocation. Analysis of the ATPase inactivation patterns revealed target sizes of 384kD and 495kD for ATP hydrolysis in native and solubilized tonoplast respectively and 430kD for H + -transport. These results suggest that the minimum size for hydrolytic or transport functions is relatively constant for the ATPase

  8. The Wheat E Subunit of V-Type H+-ATPase Is Involved in the Plant Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    Xiao-Hong Zhang

    2014-09-01

    Full Text Available The vacuolar type H+-ATPase (V-type H+-ATPase plays important roles in establishing an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.

  9. Nitric oxide and Na,K-ATPase activity in rat skeletal muscle.

    Science.gov (United States)

    Juel, C

    2016-04-01

    It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. The study used isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles, but had no effect in oxidative muscles. Spermine NONOate increased the maximal Na,K-ATPase activity by 58% (P Na,K-ATPase α-isoform. Incubation with cGMP (1 mm) increased the maximal Na,K-ATPase activity in homogenates from glycolytic muscle by 16% (P Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely, the NO/cGMP/protein kinase G signalling pathway is involved. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  10. Purification and functional motifs of the recombinant ATPase of orf virus.

    Science.gov (United States)

    Lin, Fong-Yuan; Chan, Kun-Wei; Wang, Chi-Young; Wong, Min-Liang; Hsu, Wei-Li

    2011-10-01

    Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

    Science.gov (United States)

    Iswari, S.; Palta, Jiwan P.

    1989-01-01

    Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

  12. Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle

    International Nuclear Information System (INIS)

    Bowman, B.J.; Berenski, C.J.; Jung, C.Y.

    1985-01-01

    Using radiation inactivation, the authors have measured the size of the H + -ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60 Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H + -ATPase in situ was found to be approximately 2.3 X 10(5) daltons. They also used radiation inactivation to measure the size of the Ca 2+ -ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, they directly compared the sizes of these two ATPases and found them to be essentially the same. The authors conclude that both H + -ATPase and Ca 2+ -ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides

  13. Influence of hexavanadates on Na+/K+- ATPase activity

    Directory of Open Access Journals (Sweden)

    Zdravković Aleksandra

    2016-01-01

    Full Text Available Introduction: There is a great interest in use of polioximetalates in clinical medicine, primary as antibacterial, antiviral and antitumoral agents. Considering the key role of Na+/ K+- ATPase in normal functioning of most animal cells, as well as pivotal roles in cancer cell migration, the aim of this paper was to examine the influence of new synthesized hexavandates [V6-CH3][Na]2, [V6-NO2][TBA]2, [V6-C3][H]2, [V6-C5d][TBA]2 on Na+/K+- ATPase activity. Material and methods: The enzymatic activity of porcine cerebral cortex Na+/K+- ATPase was followed in both the absence and presence of increasing concentration of [V6-CH3] [Na]2, [V6-NO2][TBA]2, [V6-C3][H]2, [V6-C5d][TBA]2 (within the range 10-8 - 10-3 mol/L. The released Pi, liberated from the enzymatic hydrolysis of ATP, was determined by spectrophotometric method, using Perkin Elmer Lambda 35 UV-VIS spectrophotometer. Results: Investigated compounds inhibit the activity of Na+/K+ ATPase in dose-dependent manner within the investigated range. Obtained results indicate that all investigated compounds inhibit the Na+/K+ ATPase activity, but with different inhibiting power. [V6-NO2] [TBA]2 (IC50 = 1,87 × 10-5 mol/L was the most potent inhibitor of Na+/K+ ATPase, while [V6-C5d][TBA]2 showed the least potent inhibiting power (IC50 = 1,31 × 10-4 mol/L . The results are consistent with previously published concentration-dependent inhibitory effect of polyoxometalates (including polioxovandates on ATPase activity from different model syistems. Conclusion: Based on the results, we can conclude that the examined compounds inhibit Na+/K+- ATPase activity in a dose-dependent manner. Inhibiting power of tested hexavanadates are different, and weaker than inhibiting power of decavanadates (tested earlier on Na+/K+- ATPase activity, which is probably due to differences in charge, size and shape of these polioxometalates. Considering the role of this enzymes in the functioning of healthy cells and the

  14. Functional Analysis of P4-ATPases

    DEFF Research Database (Denmark)

    Theorin, Lisa

    and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...... for purification of the complex by one-step purification. The ATPase activity of the ALA2/ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Changes in the phosphatidylserine headgroup or alteration of the stereochemistry affected enzymatic activity. The results demonstrate that ALA2...... is specific for phosphatidylserine and that binding of the lipid to the substrate binding site requires a unique spatial configuration of the lipid head group. Detailed information on the substrate requirements lead the way towards the full function and transport pathway of lipid flippases in plants. Recent...

  15. Stochastic Four-State Mechanochemical Model of F1-ATPase

    International Nuclear Information System (INIS)

    Wu Weixia; Zhan Yong; Zhao Tongjun; Han Yingrong; Chen Yafei

    2010-01-01

    F 1 -ATPase, a part of ATP synthase, can synthesize and hydrolyze ATP moleculars in which the central γ-subunit rotates inside the α 3 β 3 cylinder. A stochastic four-state mechanochemical coupling model of F 1 -ATPase is studied with the aid of the master equation. In this model, the ATP hydrolysis and synthesis are dependent on ATP, ADP, and Pi concentrations. The effects of ATP concentration, ADP concentration, and the external torque on the occupation probability of binding-state, the rotation rate and the diffusion coefficient of F 1 -ATPase are investigated. Moreover, the results from this model are compared with experiments. The mechanochemical mechanism F 1 -ATPase is qualitatively explained by the model. (general)

  16. Regulation of alpha1 Na/K-ATPase expression by cholesterol.

    Science.gov (United States)

    Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

    2011-04-29

    We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.

  17. Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars.

    Science.gov (United States)

    Corcoran, B M; Stanton, C; Fitzgerald, G F; Ross, R P

    2005-06-01

    Lactobacillus rhamnosus GG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose on L. rhamnosus GG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log10-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with other Lactobacillus strains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhanced L. rhamnosus GG survival from 6.4 to 8 log10 CFU ml(-1) in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition of N',N'-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F0F1-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F0F1-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 x 10(6)-fold compared with the survival of the wild type (which had a viability of 8.02 log10 CFU ml(-1)). L. rhamnosus GG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log10 CFU ml(-1) in the presence of glucose. The data indicate that glucose provides ATP to F0F1-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.

  18. ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator.

    Science.gov (United States)

    Ranjan, Rajeev; Priyanka, B S; Thakur, M S

    2014-07-01

    The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5-10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

  19. LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH

    Science.gov (United States)

    Dendy, Leslie A.; Deter, Russell L.; Philpott, Charles W.

    1973-01-01

    In an effort to determine the subcellular localization of sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) in the pseudobranch of the pinfish Lagodon rhomboides, this tissue was fractionated by differential centrifugation and the activities of several marker enzymes in the fractions were measured. Cytochrome c oxidase was found primarily in the mitochondrial-light mitochondrial (M+L) fraction. Phosphoglucomutase appeared almost exclusively in the soluble (S) fraction. Monoamine oxidase was concentrated in the nuclear (N) fraction, with a significant amount also in the microsomal (P) fraction but little in M+L or S. Na+, K+-ATPase and ouabain insensitive Mg2+-ATPase were distributed in N, M+L, and P, the former having its highest specific activity in P and the latter in M+L. Rate sedimentation analysis of the M+L fraction indicated that cytochrome c oxidase and Mg2+-ATPase were associated with a rapidly sedimenting particle population (presumably mitochondria), while Na+, K+-ATPase was found primarily in a slowly sedimenting component. At least 75% of the Na+, K+-ATPase in M+L appeared to be associated with structures containing no Mg2+-ATPase. Kinetic properties of the two ATPases were studied in the P fraction and were typical of these enzymes in other tissues. Na+, K+-ATPase activity was highly dependent on the ratio of Na+ and K+ concentrations but independent of absolute concentrations over at least a fourfold range. PMID:4349221

  20. The Na(+) cycle in Acetobacterium woodii: identification and characterization of a Na(+) translocating F(1)F(0)-ATPase with a mixed oligomer of 8 and 16 kDa proteolipids.

    Science.gov (United States)

    Müller, V; Aufurth, S; Rahlfs, S

    2001-05-01

    The homoacetogenic bacterium Acetobacterium woodii relies on a sodium ion current across its cytoplasmic membrane for energy-dependent reactions. The sodium ion potential is established by a yet to be identified primary, electrogenic pump connected to the Wood-Ljungdahl pathway. Reactions possibly involved in Na(+) export are discussed. The electrochemical sodium ion potential generated is used to drive endergonic reactions such as flagellar rotation and ATP synthesis. Biochemical and molecular data identified the Na(+)-ATPase of A. woodii as a typical member of the F(1)F(0) class of ATPases. Its catalytic properties and the hypothetical sodium ion binding site in subunit c are discussed. The encoding genes were cloned and, surprisingly, the atp operon was shown to contain multiple copies of genes encoding subunit c. Two copies encode identical 8 kDa proteolipids, and a third copy arose by duplication and subsequent fusion of two genes. Furthermore, the duplicated subunit c does not contain the ion binding site in hair pin two. Biochemical and molecular data revealed that all three copies of subunit c constitute a mixed oligomer. The evolution of the structure and function of subunit c in ATPases from eucarya, bacteria, and archaea is discussed.

  1. Activity determination of Na+ K+ - ATPase and Mg++ - ATPase enzymes in the gill of Poecilia vivipara (Osteichthyes, Cyprinodontiformes in different salinities

    Directory of Open Access Journals (Sweden)

    Marcelo da Cunha Amaral

    2001-03-01

    Full Text Available This work aimed to know the tolerance mechanisms through the salinity variation by Na+ K+ - ATPase and Mg++ - ATPase and enzymes encountered in the gills of Poecilia vivipara. In field, the presence of this species was observed in salinities of 0 and 28‰. In laboratory, these fish were maintained in aquarium with mean salinity of 30‰ and positive responses were obtained. Some adult specimens, collected in a lagoon of the Coqueiros Beach, were utilized as matrixes. In the experiments the specimens used were those born in the test aquarium. For each salinity studied three replicates were made with three specimens for each one. The alevins were maintained in salinities of 5, 10, 15, 20, 25, 30 and 35‰ during a month for adaptation. Gills were extracted in appropriate buffer for isolation of plasma membrane and used for specific dosage of the total enzymatic activity of Na+ K+ - ATPase and Mg++ - ATPase. The relation of alevins to their adaptation towards the salinity variation was also studied. The activity of the two enzymes showed a different result. The major expression of Na+ K+ - ATPase was observed in 20‰ (35 µmoles Pi.mg protein.h-1, the best salinity to cultivate P. vivipara.Este trabalho teve como objetivo conhecer os mecanismos de tolerância às variações de salinidade, pelas enzimas Mg++ - ATPase e Na+ K+ - ATPase, encontrada nas brânquias de Poecilia vivipara. No campo, foi observada a presença desta espécie em salinidades entre 0 e 28‰. No laboratório, os indivíduos foram mantidos em salinidade de 30‰ e responderam positivamente. Os indivíduos adultos, coletados em uma lagoa na praia dos Coqueiros, foram utilizados como matrizes. Nos experimentos foram usados alevinos que nasceram nos aquários testes. Para cada salinidade estudada foram feitas três réplicas com três espécimens em cada uma. Os alevinos foram mantidos em salinidades de 5, 10, 15, 20, 25, 30 e 35‰, durante um mês para total adaptação. As br

  2. Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

    Science.gov (United States)

    Ordonez, Heather; Shuman, Stewart

    2013-05-17

    We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

  3. H(+) -ATPase-defective variants of Lactobacillus delbrueckii subsp. bulgaricus contribute to inhibition of postacidification of yogurt during chilled storage.

    Science.gov (United States)

    Wang, Xinhui; Ren, Hongyang; Liu, Dayu; Wang, Bing; Zhu, Wenyou; Wang, Wei

    2013-02-01

    Continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt is the major cause of postacidification, resulting in a short shelf life. Two H(+) -ATPase defective variants of L. delbrueckii subsp. bulgaricus were successfully isolated and their H(+) -ATPase activities were reduced by 51.3% and 34.3%, respectively. It was shown that growth and acid production of variants were remarkably inhibited. The variants were more sensitive to acidic condition and had a significant rate for inactivation of H(+) -ATPase by N, N-dicyclohexylcarbodiimide (DCCD), along with a low H(+) -extrusion, suggesting that H(+) -ATPase is direct response for H(+) -extrusion. In addition, the variants were also more sensitive to NaCl, while H(+) -ATPase activities of variants and parent strain were significantly enhanced by NaCl stress. Obviously, H(+) -ATPase might be involved in Na(+) transportation. Furthermore, variants were inoculated in fermented milk to ferment yogurt. There was no significant difference in flavor, whereas the postacidification of yogurt during chilled storage was remarkably inhibited. It is suggested that application of L. delbrueckii subsp. bulgaricus with reduced H(+) -ATPase activity in yogurt fermentation is one of effect, economic and simple avenues of inhibiting postacidification of yogurt during refrigerated storage, giving a longer shelf life. During yogurt fermentation, continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt leads to milk fermentation with high postacidification, resulting in a short shelf life. In this work, 2 acid-sensitive variant strains of L. delbrueckii subsp. bulgaricus were isolated. The characteristics related to H(+) -ATPase were compared and it was observed that milk fermented by the variants had lower postacidification, giving a longer shelf life. Application of L. delbrueckii subsp. bulgaricus with reduced H(+) -ATPase activity

  4. Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles and an alternative pathway with DMSO

    Directory of Open Access Journals (Sweden)

    Marilena Carbone

    2016-03-01

    Full Text Available Proton caged compounds exhibit a characteristic behavior when directly dosed into cells or being coupled to gold nanoparticles prior to the dosing. When irradiated in the near ultraviolet region, they release protons that interact with intracellular HCO3− to yield H2CO3. The dissociation of carbonic acid, then, releases CO2 that can be distinctively singled out in infrared spectra.In the process of searching a pathway to augment the intracellular uptake of proton caged compounds, we probed the association of 1-(2-nitrophenyl-ethylhexadecyl sulfonate (HDNS with DMSO, an agent to enhance the membrane permeability. We found out a different UV-induced protonation mechanism that opens up to new conduits of employing of proton caged compounds. Here, we report the infrared data we collected in this set of experiments. Keywords: Proton caged compounds, DMSO, Intracellular proton release

  5. Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

    Science.gov (United States)

    Qiu, Li Yan; Krieger, Elmar; Schaftenaar, Gijs; Swarts, Herman G P; Willems, Peter H G M; De Pont, Jan Joep H H M; Koenderink, Jan B

    2005-09-16

    Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

  6. The effect of different doses of epidermal growth factor on liver ornithine decarboxylase and Na-K ATPase activities in newborn rats.

    Science.gov (United States)

    Bilgihan, A; Turkozkan, N; Isman, F; Kilinc, M; Demirsoy, S

    1998-08-01

    1. Ornithine decarboxylase and Na-K ATPase activities were studied in rat livers that were treated with different doses of epidermal growth factor (EGF). 2. The ornithine decarboxylase activities were studied with spectrophotometry, and results were expressed as micromoles of putrescine per hour per milligram of protein. Na-K ATPase activities were studied on the basis of the principle of measuring the amount of inorganic phosphates released by the hydrolysis of ATP, and the results were expressed as micromoles of inorganic phosphate per hour per milligram of protein. 3. When compared with the controls, although the Na-K ATPase activities were decreased at low doses of EGF, their activities were found to be increased at high doses of EGF. On the other hand, there was a positive correlation between ornithine decarboxylase activities and EGF doses. 4. The results of this study suggest that, whereas the decrease in Na-K ATPase activities at low doses of EGF can be due to the utilization of the enzyme, the increase in Na-K ATPase activities at high doses of EGF can be attributed to its enhanced synthesis.

  7. Uptake and translocation of imidacloprid, clothianidin and flupyradifurone in seed-treated soybeans.

    Science.gov (United States)

    Stamm, Mitchell D; Heng-Moss, Tiffany M; Baxendale, Frederick P; Siegfried, Blair D; Blankenship, Erin E; Nauen, Ralf

    2016-06-01

    Seed treatment insecticides have become a popular management option for early-season insect control. This study investigated the total uptake and translocation of seed-applied [(14) C]imidacloprid, [(14) C]clothianidin and [(14) C]flupyradifurone into different plant parts in three soybean vegetative stages (VC, V1 and V2). The effects of soil moisture stress on insecticide uptake and translocation were also assessed among treatments. We hypothesized that (1) uptake and translocation would be different among the insecticides owing to differences in water solubility, and (2) moisture stress would increase insecticide uptake and translocation. Uptake and translocation did not follow a clear trend in the three vegetative stages. Initially, flupyradifurone uptake was greater than clothianidin uptake in VC soybeans. In V1 soybeans, differences in uptake among the three insecticides were not apparent and unaffected by soil moisture stress. Clothianidin was negatively affected by soil moisture stress in V2 soybeans, while imidacloprid and flupyradifurone were unaffected. Specifically, soil moisture stress had a positive effect on the distribution of flupyradifurone in leaves. This was not observed with the neonicotinoids. This study enhances our understanding of the uptake and distribution of insecticides used as seed treatments in soybean. The uptake and translocation of these insecticides differed in response to soil moisture stress. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  8. Radiation dose of aircrews during a solar proton event without ground-level enhancement

    Directory of Open Access Journals (Sweden)

    R. Kataoka

    2015-01-01

    Full Text Available A significant enhancement of radiation doses is expected for aircrews during ground-level enhancement (GLE events, while the possible radiation hazard remains an open question during non-GLE solar energetic particle (SEP events. Using a new air-shower simulation driven by the proton flux data obtained from GOES satellites, we show the possibility of significant enhancement of the effective dose rate of up to 4.5 μSv h−1 at a conventional flight altitude of 12 km during the largest SEP event that did not cause a GLE. As a result, a new GOES-driven model is proposed to give an estimate of the contribution from the isotropic component of the radiation dose in the stratosphere during non-GLE SEP events.

  9. The AAA+ ATPase p97, a cellular multitool.

    Science.gov (United States)

    Stach, Lasse; Freemont, Paul S

    2017-08-17

    The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy. © 2017 The Author(s).

  10. A plasma membrane H + ATPase gene is germinationinduced in ...

    African Journals Online (AJOL)

    A plasma membrane H + ATPase gene is germinationinduced in wheat embryos. ... African Journal of Biotechnology ... of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods.

  11. Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

    Science.gov (United States)

    Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

    2016-10-18

    The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the

  12. Dietary acidification enhances phosphorus digestibility but decreases H+/K+-ATPase expression in rainbow trout.

    Science.gov (United States)

    Sugiura, Shozo H; Roy, Prabir K; Ferraris, Ronaldo P

    2006-10-01

    Oxynticopeptic cells of fish stomach are thought to secrete less acid than the specialized parietal cells of mammalian stomach. Gastric acidity, however, has not been directly compared between fish and mammals. We therefore fed rainbow trout and rats the same meal, and found that the lowest postprandial pH of trout stomach was 2.7, which was only transiently sustained for 1 h, whereas that of rat stomach was 1.3, which was sustained for 3 h. Postprandial pH of the small intestine was slightly higher in trout (approximately 8.0) than in rats (approximately 7.6), but pH of the large intestine was similar (approximately 8.0). Addition of acids to fish feeds, in an attempt to aid the weak acidity of fish stomach, has been known to improve phosphorus digestibility, but its physiological effect on fish stomach is not known. Exogenous acids did improve phosphorus digestibility but also decreased steady-state mRNA expression of trout H(+)/K(+)-ATPase (ATP4A, the proton pump) as well as Na(+)/bicarbonate cotransporter (NBC), and had no effect on gastrin-like mRNA and somastostatin (SST) mRNA abundance. Gastrin-like mRNA and SST-2 mRNA were equally distributed between corpus and antrum. ATP4A mRNA and NBC mRNA were in the corpus, whereas SST-1 mRNA was in the antrum. Trout gastrin-like EST had modest homology to halibut and pufferfish gastrin, whereas trout ATP4A mRNA had > or = 95% amino acid homology with mammalian, Xenopus and flounder ATP4A. Although ATP4A seems highly conserved among vertebrates, gastric acidity is much less in trout than in rats, explaining the low digestibility of bone phosphorus, abundant in fish diets. Dietary acidification does not reduce acidity enough to markedly improve phosphorus digestibility, perhaps because exogenous acids may inhibit endogenous acid production.

  13. Northern Hemisphere Atmospheric Influence of the Solar Proton Events and Ground Level Enhancement in January 2005

    Science.gov (United States)

    Jackman, C. H.; Marsh, D. R.; Vitt, F. M.; Roble, R. G.; Randall, C. E.; Bernath, P. F.; Funke, B.; Lopez-Puertas, M.; Versick, S.; Stiller, G. P.; hide

    2011-01-01

    Solar eruptions in early 2005 led substantial barrage of charged particles on the Earth's atmosphere during the January 16-21 period. Proton fluxes were greatly increased during these several days and led to the production ofHO(x)(H, OH, BO2)and NO(x)(N, NO, NO2), which then caused the destruction of ozone. We focus on the Northern polar region, where satellite measurements and simulations with the Whole Atmosphere Community Climate Model (WACCM3) showed large enhancements in mesospheric HO(x) and NO(x) constituents, and associated ozone reductions, due 10 these solar proton events (SPEs). The WACCM3 simulations show enhanced short-lived OH throughout the mesosphere in the 60-82.5degN latitude band due to the SPEs for most days in the Jan.16-2l,2005 period, in reasonable agreement with the Aura Microwave Limb Sounder (MLS) measurements. Mesospheric HO2 is also predicted to be increased by the SPEs, however, the modeled HO2 results are somewhat larger than the MLS measurements. These HO(x) enhancements led to huge predicted and MLS-measured ozone decreases of greater than 40% throughout most of the Northern polar mesosphere during the SPE period. Envisat Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) measurements of hydrogen peroxide (H2O2) show increases throughout the stratosphere with highest enhancements of about 60 ppt y in the lowermost mesosphere over the Jan. 16-18, 2005 period due to the solar protons. WACCM3 predictions indicate H2O2 enhancements over the same time period of more than twice that amount. Measurements of nitric acid (HNO3) by both MLS and MIPAS show an increase of about 1 ppbv above background levels in the upper stratosphere during January 16-29, 2005. WACCM3 simulations show only minuscule HNO3 changes in the upper stratosphere during this time period. However due to the small loss rates during winter, polar mesospheric enhancements of NO(x) are computed to be greater than 50 ppbv during the SPE period. Computed NO

  14. Northern Hemisphere atmospheric influence of the solar proton events and ground level enhancement in January 2005

    Directory of Open Access Journals (Sweden)

    C. H. Jackman

    2011-07-01

    Full Text Available Solar eruptions in early 2005 led to a substantial barrage of charged particles on the Earth's atmosphere during the 16–21 January period. Proton fluxes were greatly increased during these several days and led to the production of HOx (H, OH, HO2 and NOx (N, NO, NO2, which then caused the destruction of ozone. We focus on the Northern polar region, where satellite measurements and simulations with the Whole Atmosphere Community Climate Model (WACCM3 showed large enhancements in mesospheric HOx and NOx constituents, and associated ozone reductions, due to these solar proton events (SPEs. The WACCM3 simulations show enhanced short-lived OH and HO2 concentrations throughout the mesosphere in the 60–82.5° N latitude band due to the SPEs for most days in the 16–21 January 2005 period, somewhat higher in abundance than those observed by the Aura Microwave Limb Sounder (MLS. These HOx enhancements led to huge predicted and MLS-measured ozone decreases of greater than 40 % throughout most of the northern polar mesosphere during the SPE period. Envisat Michelson Interferometer for Passive Atmospheric Sounding (MIPAS measurements of hydrogen peroxide (H2O2 show increases throughout the stratosphere with highest enhancements of about 60 pptv in the lowermost mesosphere over the 16–18 January 2005 period due to the solar protons. WACCM3 predictions indicate H2O2 enhancements over the same time period of about three times that amount. Measurements of nitric acid (HNO3 by both MLS and MIPAS show an increase of about 1 ppbv above background levels in the upper stratosphere during 16–29 January 2005. WACCM3 simulations show only minuscule HNO3 increases (<0.05 ppbv in the upper stratosphere during this time period. Polar mesospheric enhancements of NOx are computed to be greater than 50

  15. Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2.

    Science.gov (United States)

    Vestergaard, Anna L; Coleman, Jonathan A; Lemmin, Thomas; Mikkelsen, Stine A; Molday, Laurie L; Vilsen, Bente; Molday, Robert S; Dal Peraro, Matteo; Andersen, Jens Peter

    2014-04-08

    P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.

  16. Tuning of Nafion® by HKUST-1 as coordination network to enhance proton conductivity for fuel cell applications

    Science.gov (United States)

    Kim, Hee Jin; Talukdar, Krishan; Choi, Sang-June

    2016-02-01

    Metal-organic frameworks can be intentionally coordinated to achieve improved proton conductivity because they have highly ordered structures and modular nature that serve as a scaffold to anchor acidic groups and develop efficient proton transfer pathways for fuel cell application. Using the concept of a coordination network, the conductivity of Nafion® was tuned by the incorporation of HKUST-1. It has CuII-paddle wheel type nodes and 1,3,5-benzenetricarboxylate struts, feature accessible sites that provides an improved protonic channel depending on the water content. In spite of the fact that HKUST-1 is neutral, coordinated water molecules are contributed adequately acidic by CuII to supply protons to enhance proton conductivity. Water molecules play a vital part in transfer of proton as conducting media and serve as triggers to change proton conductivity through reforming hydrogen bonding networks by water adsorption/desorption process. Increased ion exchange capacity and proton conductivity with lower water uptake of the H3PO4-doped material, and improved thermal stability (as confirmed by thermogravimetric analysis) were achieved. The structure of HKUST-1 was confirmed via field emission scanning electron microscopy and X-ray diffraction, while the porosity and adsorption desorption capacity were characterized by porosity analysis.

  17. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase...

  18. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg

    2010-01-01

    pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties...

  19. Markovian description of unbiased polymer translocation

    International Nuclear Information System (INIS)

    Mondaini, Felipe; Moriconi, L.

    2012-01-01

    We perform, with the help of cloud computing resources, extensive Langevin simulations which provide compelling evidence in favor of a general Markovian framework for unbiased three-dimensional polymer translocation. Our statistical analysis consists of careful evaluations of (i) two-point correlation functions of the translocation coordinate and (ii) the empirical probabilities of complete polymer translocation (taken as a function of the initial number of monomers on a given side of the membrane). We find good agreement with predictions derived from the Markov chain approach recently addressed in the literature by the present authors. -- Highlights: ► We investigate unbiased polymer translocation through membrane pores. ► Large statistical ensembles have been produced with the help of cloud computing resources. ► We evaluate the two-point correlation function of the translocation coordinate. ► We evaluate empirical probabilities for complete polymer translocation. ► Unbiased polymer translocation is described as a Markov stochastic process.

  20. Markovian description of unbiased polymer translocation

    Energy Technology Data Exchange (ETDEWEB)

    Mondaini, Felipe [Instituto de Física, Universidade Federal do Rio de Janeiro, C.P. 68528, 21945-970 Rio de Janeiro, RJ (Brazil); Centro Federal de Educação Tecnológica Celso Suckow da Fonseca, UnED Angra dos Reis, Angra dos Reis, 23953-030, RJ (Brazil); Moriconi, L., E-mail: moriconi@if.ufrj.br [Instituto de Física, Universidade Federal do Rio de Janeiro, C.P. 68528, 21945-970 Rio de Janeiro, RJ (Brazil)

    2012-10-01

    We perform, with the help of cloud computing resources, extensive Langevin simulations which provide compelling evidence in favor of a general Markovian framework for unbiased three-dimensional polymer translocation. Our statistical analysis consists of careful evaluations of (i) two-point correlation functions of the translocation coordinate and (ii) the empirical probabilities of complete polymer translocation (taken as a function of the initial number of monomers on a given side of the membrane). We find good agreement with predictions derived from the Markov chain approach recently addressed in the literature by the present authors. -- Highlights: ► We investigate unbiased polymer translocation through membrane pores. ► Large statistical ensembles have been produced with the help of cloud computing resources. ► We evaluate the two-point correlation function of the translocation coordinate. ► We evaluate empirical probabilities for complete polymer translocation. ► Unbiased polymer translocation is described as a Markov stochastic process.

  1. Structural and functional studies of a Cu+-ATPase from Legionella pneumophila

    DEFF Research Database (Denmark)

    Mattle, Daniel

    During his studies, Daniel Mattle explored the copper(I) export mechanism of a P-type Cu+ ATPase from Legionella pneumophila – a homologue to the human Cu+ ATPases. Cu+ ATPases are responsible for the homeostatic control of the physiological relevant – but toxic – copper(I) cations. To assess...

  2. Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Vardanyan, Zaruhi [Department of Biophysics of the Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan (Armenia); Trchounian, Armen, E-mail: trchounian@ysu.am [Department of Biophysics of the Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan (Armenia)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fe{sup 3+} stimulates but Fe{sup 2+} suppresses Enterococcus hirae wild-type and atpD mutant growth. Black-Right-Pointing-Pointer Fe ions change oxidation-reduction potential drop during cell growth. Black-Right-Pointing-Pointer Fe{sup 3+} and Fe{sup 2+} have opposite effects on a membrane-associated ATPase activity. Black-Right-Pointing-Pointer These effects are either in the presence of F{sub 0}F{sub 1} inhibitor or non-functional F{sub 0}F{sub 1}. Black-Right-Pointing-Pointer Fe ions decrease protons and coupled potassium ions fluxes across the membrane. -- Abstract: Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E{sub h}) from positive values to negative ones (down to {approx}-200 mV). In this study, iron (III) ions (Fe{sup 3+}) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe{sup 2+}) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E{sub h} values during bacterial growth. It was revealed that ATPase activity with and without N,N Prime -dicyclohexylcarbodiimide (DCCD), an inhibitor of the F{sub 0}F{sub 1}-ATPase, increased in the presence of even low Fe{sup 3+} concentration (0.05 mM) but decreased in the presence of Fe{sup 2+}. It was established that Fe{sup 3+} and Fe{sup 2+} both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe{sup 2+} with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F{sub 0}F{sub 1}) MS116 strains but they were different with Fe{sup 3+} and Fe{sup 2+}. It is suggested that the effects of Fe{sup 3+} might be due to

  3. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  4. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

    2009-01-01

    ,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

  5. Kidney in potassium depletion. I. Na/sup +/-K/sup +/-ATPase activity and (/sup 3/H)ouabain binding in MCT

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, M.; Katz, A.I.

    1987-03-01

    The effect of potassium depletion on renal Na/sup +/K/sup +/-ATPase was studied in rats. K depletion produced a striking, time-dependent increase in Na/sup +/-K/sup +/-ATPase activity of the outer medullary collecting tubules (inner stripe; MCT/sub is/). After 3 wk on the K-free diet, when the urine was almost potassium-free, Na/sup +/-K/sup +/-ATPase activity in MCT/sub is/ was over fourfold higher than in control animals. Repletion of potassium restored enzyme activity to base line within 7 days which corresponds to the catabolic rate of the renal enzyme, suggesting the cessation of enhanced synthesis that took place during K deprivation. Changes in Na/sup +/-K/sup +/-ATPase activity and aldosterone levels during both K depletion and repletion occurred in opposite directions and were therefore independent of each other. (/sup 3/H)Ouabain binding to intact MCT/sub is/, reflecting the number of pump sites on the basolateral membrane, was similar in K-depleted and control animals; in contrast, tubule permeabilization that exposes additional pump units to the ligand, unmasked a nearly fourfold increase in (/sup 3/H)ouabain binding in K-depleted rats, comparable to the increment in Na/sup +/-K/sup +/-ATPase activity. These results show that K depletion leads to a marked increase in Na/sup +/-K/sup +/-ATPase activity of MCT/sub is/, and suggest that the new enzyme units are located at a ouabain-inaccessible site in the intact tubule, i.e., either in an intracellular compartment or at the luminal membrane, where they may be involved in potassium reabsorption.

  6. Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

    International Nuclear Information System (INIS)

    Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

    1987-01-01

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

  7. Radioprotector modifying influence upon the ion transport ATPase activities

    International Nuclear Information System (INIS)

    Dvoretsky, A.I.; Egorova, E.G.; Ananieva, T.V.; Kulikova, I.A.

    1993-01-01

    The effects of aminothiol and biogenic amine radioprotectors (β-mercaptoethylamine, AET, serotonin, dopamine, histamine) on the basic ion transport enzymes, such as Na, K-ATP ase and Mg, Ca-ATPase activities were investigated in the tissues of numerous organs, with different radiosensitivity in the wistar rats. Experimental results showed that intraperitoneal injection of the used radioprotectors caused preliminary inhibition of the Na, K-ATPase activity in tissues from organs with different radioresistance, but had no influence on the Mg, Ca-ATPase activity in membranes of erythrocytes and rat brain cells. (2 tabs.)

  8. Spot Scanning and Passive Scattering Proton Therapy: Relative Biological Effectiveness and Oxygen Enhancement Ratio in Cultured Cells.

    Science.gov (United States)

    Iwata, Hiromitsu; Ogino, Hiroyuki; Hashimoto, Shingo; Yamada, Maho; Shibata, Hiroki; Yasui, Keisuke; Toshito, Toshiyuki; Omachi, Chihiro; Tatekawa, Kotoha; Manabe, Yoshihiko; Mizoe, Jun-etsu; Shibamoto, Yuta

    2016-05-01

    To determine the relative biological effectiveness (RBE), oxygen enhancement ratio (OER), and contribution of the indirect effect of spot scanning proton beams, passive scattering proton beams, or both in cultured cells in comparison with clinically used photons. The RBE of passive scattering proton beams at the center of the spread-out Bragg peak (SOBP) was determined from dose-survival curves in 4 cell lines using 6-MV X rays as controls. Survival of 2 cell lines after spot scanning and passive scattering proton irradiation was then compared. Biological effects at the distal end region of the SOBP were also investigated. The OER of passive scattering proton beams and 6 MX X rays were investigated in 2 cell lines. The RBE and OER values were estimated at a 10% cell survival level. The maximum degree of protection of radiation effects by dimethyl sulfoxide was determined to estimate the contribution of the indirect effect against DNA damage. All experiments comparing protons and X rays were made under the same biological conditions. The RBE values of passive scattering proton beams in the 4 cell lines examined were 1.01 to 1.22 (average, 1.14) and were almost identical to those of spot scanning beams. Biological effects increased at the distal end of the SOBP. In the 2 cell lines examined, the OER was 2.74 (95% confidence interval, 2.56-2.80) and 3.08 (2.84-3.11), respectively, for X rays, and 2.39 (2.38-2.43) and 2.72 (2.69-2.75), respectively, for protons (Pcells between X rays and protons). The maximum degree of protection was significantly higher for X rays than for proton beams (P<.05). The RBE values of spot scanning and passive scattering proton beams were almost identical. The OER was lower for protons than for X rays. The lower contribution of the indirect effect may partly account for the lower OER of protons. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

    Science.gov (United States)

    Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

    2009-01-01

    A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

  10. Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    Li Chunrong; Iida, Mari; Dunn, Emily F.; Wheeler, Deric L.

    2010-01-01

    Background and purpose: The aberrant expression of epidermal growth factor receptor (EGFR) has been linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III trial combining cetuximab with radiation confirmed a strong survival advantage. However, both cetuximab and radiation can promote EGFR translocation to the nucleus where it enhances resistance to both of these modalities. In this report we sought to determine how to block cetuximab- and radiation-induced translocation of EGFR to the nucleus in HNSCC cell lines. Material and methods: We utilized three established HNSCC cell lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of EGFR after treatment with cetuximab or radiation. We then utilized dasatinib (BMS-354825), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases, to determine if SFKs blockade could abrogate cetuximab- and radiation-induced nuclear EGFR translocation. Results: Cetuximab and radiation treatment of all three HNSCC lines lead to translocation of the EGFR to the nucleus. Blockade of SFKs abrogated cetuximab- and radiation-induced EGFR translocation to the nucleus. Conclusions: The data presented in this report suggest that both cetuximab and radiation can promote EGFR translocation to the nucleus and dasatinib can inhibit this process. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC.

  11. Suitability of amphibians and reptiles for translocation.

    Science.gov (United States)

    Germano, Jennifer M; Bishop, Phillip J

    2009-02-01

    Translocations are important tools in the field of conservation. Despite increased use over the last few decades, the appropriateness of translocations for amphibians and reptiles has been debated widely over the past 20 years. To provide a comprehensive evaluation of the suitability of amphibians and reptiles for translocation, we reviewed the results of amphibian and reptile translocation projects published between 1991 and 2006. The success rate of amphibian and reptile translocations reported over this period was twice that reported in an earlier review in 1991. Success and failure rates were independent of the taxonomic class (Amphibia or Reptilia) released. Reptile translocations driven by human-wildlife conflict mitigation had a higher failure rate than those motivated by conservation, and more recent projects of reptile translocations had unknown outcomes. The outcomes of amphibian translocations were significantly related to the number of animals released, with projects releasing over 1000 individuals being most successful. The most common reported causes of translocation failure were homing and migration of introduced individuals out of release sites and poor habitat. The increased success of amphibian and reptile translocations reviewed in this study compared with the 1991 review is encouraging for future conservation projects. Nevertheless, more preparation, monitoring, reporting of results, and experimental testing of techniques and reintroduction questions need to occur to improve translocations of amphibians and reptiles as a whole.

  12. Receptor kinase-mediated control of primary active proton pumping at the plasma membrane

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Kristensen, Astrid; Cuin, Tracey A.

    2014-01-01

    Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro...... and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast...... heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2...

  13. Cell-based lipid flippase assay employing fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Günther-Pomorski, Thomas

    2016-01-01

    P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, s...... flippase activities in the plasma membrane of cells, using yeast as an example.......P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases......, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid...

  14. Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

    Science.gov (United States)

    de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

    1995-05-01

    Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

  15. Role of Human Na,K-ATPase alpha 4 in Sperm Function, Derived from Studies in Transgenic Mice

    Science.gov (United States)

    McDermott, Jeffrey; Sánchez, Gladis; Nangia, Ajay K.; Blanco, Gustavo

    2014-01-01

    SUMMARY Most of our knowledge on the biological role of the testis-specific Na,K-ATPase alpha 4 isoform derives from studies performed in non-human species. Here, we studied the function of human Na,K-ATPase alpha 4 after its expression in transgenic mice. Using a bacterial artificial chromosome (BAC) construct, containing the human ATP1A4 gene locus, we obtained expression of the human α4 transgene specifically in mouse sperm, enriched in the sperm flagellum. The expressed, human alpha 4 was active, and compared to wild-type sperm, those from transgenic mice displayed higher Na,K-ATPase alpha 4 activity and greater binding of fluorescently labeled ouabain, which is typical of the alpha 4 isoform. The expression and activity of endogenous alpha 4 and the other Na,K-ATPase alpha isoform present in sperm, alpha 1, remained unchanged. Male mice expressing the human ATP1A4 transgene exhibited similar testis size and morphology, normal sperm number and shape, and no changes in overall fertility compared to wild-type mice. Sperm carrying the human transgene exhibited enhanced total motility and an increase in multiple parameters of sperm movement, including higher sperm hyperactive motility. In contrast, no statistically significant changes in sperm membrane potential, protein tyrosine phosphorylation, or spontaneous acrosome reaction were found between wild-type and transgenic mice. Altogether, these results provide new genetic evidence for an important role of human Na,K-ATPase alpha 4 in sperm motility and hyperactivation, and establishes a new animal model for future studies of this isoform. PMID:25640246

  16. Enhanced stimulation of chromosomal translocations and sister chromatid exchanges by either HO-induced double-strand breaks or ionizing radiation in Saccharomyces cerevisiae yku70 mutants

    International Nuclear Information System (INIS)

    Fasullo, Michael; St Amour, Courtney; Zeng Li

    2005-01-01

    DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous endjoining (NHEJ). In Saccharomyces cerevisiae, expression of both MAT a and MATα inhibits NHEJ and facilitates DSB-initiated HR. We previously observed that DSB-initiated recombination between two his3 fragments, his3-Δ5' and his3-Δ3'::HOcs is enhanced in haploids and diploids expressing both MAT a and MATα genes, regardless of the position or orientation of the his3 fragments. Herein, we measured frequencies of DNA damage-associated translocations and sister chromatid exchanges (SCEs) in yku70 haploid mutants, defective in NHEJ. Translocation and SCE frequencies were measured in strains containing the same his3 fragments after DSBs were made directly at trp1::his3-Δ3'::HOcs. Wild type and yku70 cells were also exposed to ionizing radiation and radiomimetic agents methyl methanesulfonate (MMS), phleomycin, and 4-nitroquinolone-1-oxide (4-NQO). Frequencies of X-ray-associated and DSB-initiated translocations were five-fold higher in yku70 mutants compared to wild type; however, frequencies of phleomycin-associated translocations were lower in the yku70 haploid mutant. Frequencies of DSB-initiated SCEs were 1.8-fold higher in the yku70 mutant, compared to wild type. Thus, DSB-initiated HR between repeated sequences on non-homologous chromosomes and sister chromatids occurs at higher frequencies in yku70 haploid mutants; however, higher frequencies of DNA damage-associated HR in yku70 mutants depend on the DNA damaging agent

  17. The role of the plasma membrane H+-ATPase in plant-microbe interactions.

    Science.gov (United States)

    Elmore, James Mitch; Coaker, Gitta

    2011-05-01

    Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant-pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility.

  18. Oxidative stress (glutathionylation and Na,K-ATPase activity in rat skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Carsten Juel

    Full Text Available Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation on the Na,K-ATPase in rat skeletal muscle membranes.Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05 in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0-10 mM increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.

  19. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Directory of Open Access Journals (Sweden)

    Huzhang Mao

    2016-03-01

    Full Text Available Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

  20. Alteration of aluminium inhibition of synaptosomal (Na(+)/K(+))ATPase by colestipol administration.

    Science.gov (United States)

    Silva, V S; Oliveira, L; Gonçalves, P P

    2013-11-01

    The ability of aluminium to inhibit the (Na(+)/K(+))ATPase activity has been observed by several authors. During chronic dietary exposure to AlCl3, brain (Na(+)/K(+))ATPase activity drops, even if no alterations of catalytic subunit protein expression and of energy charge potential are observed. The aluminium effect on (Na(+)/K(+))ATPase activity seems to implicate the reduction of interacting protomers within the oligomeric ensemble of the membrane-bound (Na(+)/K(+))ATPase. The activity of (Na(+)/K(+))ATPase is altered by the microviscosity of lipid environment. We studied if aluminium inhibitory effect on (Na(+)/K(+))ATPase is modified by alterations in synaptosomal membrane cholesterol content. Adult male Wistar rats were submitted to chronic dietary AlCl3 exposure (0.03 g/day of AlCl3) and/or to colestipol, a hypolidaemic drug (0.31 g/day) during 4 months. The activity of (Na(+)/K(+))ATPase was studied in brain cortex synaptosomes with different cholesterol contents. Additionally, we incubate synaptosomes with methyl-β-cyclodextrin for both enrichment and depletion of membrane cholesterol content, with or without 300 μM AlCl3. This enzyme activity was significantly reduced by micromolar AlCl3 added in vitro and when aluminium was orally administered to rats. The oral administration of colestipol reduced the cholesterol content and concomitantly inhibited the (Na(+)/K(+))ATPase. The aluminium inhibitory effect on synaptosomal (Na(+)/K(+))ATPase was reduced by cholesterol depletion both in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Search for Sphalerons in Proton-Proton Collisions

    CERN Document Server

    Ellis, John

    2016-04-14

    In a recent paper, Tye and Wong (TW) have argued that sphaleron-induced transitions in high-energy proton-proton collisions should be enhanced compared to previous calculations, based on a construction of a Bloch wave function in the periodic sphaleron potential and the corresponding pass band structure. Here we convolute the calculations of TW with parton distribution functions and simulations of final states to explore the signatures of sphaleron transitions at the LHC and possible future colliders. We calculate the increase of sphaleron transition rates in proton-proton collisions at centre-of-mass energies of 13/14/33/100 TeV for different sphaleron barrier heights, while recognising that the rates have large overall uncertainties. We use a simulation to show that LHC searches for microscopic black holes should have good efficiency for detecting sphaleron-induced final states, and discuss their experimental signatures and observability in Run 2 of the LHC and beyond. We recast the early ATLAS Run-2 search...

  2. Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin

    2011-10-01

    Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.

  3. Two-week inhalation of budesonide increases muscle Na,K ATPase content but not endurance in response to terbutaline in men.

    Science.gov (United States)

    Hostrup, M; Jessen, S; Onslev, J; Clausen, T; Porsbjerg, C

    2017-07-01

    While chronic systemic administration of glucocorticoids increases muscle Na + ,K + ATPase content, such effect is unexplored after therapeutic inhalation. We investigated the effect of therapeutic inhalation of the glucocorticoid budesonide on Na + ,K + ATPase content of skeletal muscle in men. Ten healthy trained subjects, aged 23 ± 4 years (mean ± 95% CI), participated in the study. Before and after 2 weeks of daily inhalation of budesonide (1.6 mg/day), a biopsy was taken from the vastus lateralis muscle for measurement of Na + ,K + ATPase content and blood samples were drawn for determination of plasma budesonide, cortisol, and K + . Subjects' performance during cycling to fatigue at 90% of incremental peak power output (iPPO) was measured in response to 4 mg inhaled terbutaline to maximally stimulate Na + ,K + ATPase activity. Plasma concentrations of budesonide rose to 5.0 ± 1.6 nM with the intervention, whereas no changes were observed in plasma cortisol. Muscle Na + ,K + ATPase content increased (P ≤ 0.01) by 46 ± 34 pmol/(g wet wt) (17% increase) with the intervention. Cycling performance at 90% of iPPO did not change (P = 0.21) with the intervention (203 vs 214 s) in response to terbutaline. The present observations show that therapeutic inhalation of glucocorticoids increases muscle Na + ,K + ATPase content, but does not enhance high-intensity cycling endurance in response to terbutaline. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Substrate Specificity of Na+,Cl-(HCO3-)-ATPase.

    Science.gov (United States)

    Yurkiv, V A; Melikhov, V I; Shubin, V S

    2016-09-01

    We studied substrate specificity of Na + ,Cl - (HCO 3 - )-ATPase. In most cases, replacement of ATP for other phosphate-containing substances resulted in not only pronounced suppression of phosphohydrolase reactions, but also dramatic changes of their responsiveness to the stimulating effect of monovalent ions. The data showed that Na + ,Cl - (HCO 3 - )-ATPase is a highly specific enzyme for ATP.

  5. The Role of Janus Kinase 3 in the Regulation of Na+/K+ ATPase under Energy Depletion

    Directory of Open Access Journals (Sweden)

    Zohreh Hosseinzadeh

    2015-05-01

    Full Text Available Background/Aims: Janus kinase-3 (JAK3 is activated during energy depletion. Energy-consuming pumps include the Na+/K+-ATPase. The present study explored whether JAK3 regulates Na+/K+-ATPase in dendritic cells (DCs. Methods: Ouabain (100 µM-sensitive (Iouabain and K+-induced (Ipump outward currents were determined by utilizing whole cell patch-clamp, Na+/K+-ATPase α1-subunit mRNA levels by RT-PCR, Na+/K+-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3-/- or wild-type mice (jak3+/+. Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active A568VJAK3 or inactive K851AJAK3. Results: Na+/K+-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3-/-DCs than in jak3+/+DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3+/+ DCs but not in jak3-/-DCs. Cellular ATP was significantly lower in jak3-/-DCs than in jak3+/+DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3-/-DCs than in jak3+/+DCs and strongly blunted by ouabain in both jak3+/+ and jak3-/-DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of A568VJAK3 but not of K851AJAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenylamino]-6,7-dimethoxyquinazoline, 22 μM enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between A568VJAK3 and K851AJAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM. Conclusions: JAK3 down-regulates Na+/K+-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

  6. Nitric oxide and Na,K-ATPase activity in rat skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten

    2016-01-01

    Aim: It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. Method: The study used...... isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Results: Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles...... activity was depressed by oxidized glutathione. Conclusion: NO and cGMP stimulate the Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely...

  7. Regulation of α1 Na/K-ATPase Expression by Cholesterol*

    OpenAIRE

    Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

    2011-01-01

    We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol tr...

  8. Electochemical detection of chromosome translocation

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dimaki, Maria; Silahtaroglu, Asli

    2014-01-01

    Cytogenetics is a study of the cell structure with a main focus on chromosomes content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders and heametological malignancies. Chromosome translocations are structural rearrangements of two...... chromosomes that results in formation of derivative chromosomes with a mixed DNA sequence. The method currently used for their detection is Fluorescent In Situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the derivative chromosomes. We present here a double...... hybridization approach developed for label-free detection of the chromosome translocations. For specific translocation detection it is necessary to determine that the two DNA sequences forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The electrochemical...

  9. Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

    Science.gov (United States)

    Liu, Lijun; Ivanov, Alexander V; Gable, Marjorie E; Jolivel, Florent; Morrill, Gene A; Askari, Amir

    2011-10-11

    To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

  10. The effect of using a "soft" release on translocation success of red-cockaded woodpeckers.

    Energy Technology Data Exchange (ETDEWEB)

    Franzreb, Kathleen, E.

    2004-12-31

    Franzreb, Kathleen, E. 2004 The effect of using a "soft" release on translocation success of red-cockaded woodpeckers. In: Red-cockaded woodpecker; Road to Recovery. Proceedings of the 4th Red-cockaded woodpecker Symposium. Ralph Costa and Susan J. Daniels, eds. Savannah, Georgia. January, 2003. Chapter 6. Translocation. Pp 301-306. Abstract: Translocations of the endangered red-cockaded woodpecker have been conducted since 1986 to enhance critically small subpopulations, to minimize the likelihood of local extirpations, and to reduce the adverse effects of fragmentation and isolation among existing populations. Such attempts have had mixed success. This article compares "hard" releases with a "soft" release technique where the birds are temporarily interned in a large aviary at the release point for a period of 9 to 14 days.

  11. Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

    Science.gov (United States)

    Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

    2002-05-01

    White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

  12. Translocation of 14C-photosynthates under normal and moisture stress conditions in finger millet (Eleusine coracana) gaertin

    International Nuclear Information System (INIS)

    Udayakumar, M.; Rama Rao, S.; Krishna Sastry, K.S.

    1981-01-01

    Translocation of photosynthates into different sinks was studied following feeding a single leaf with 14 CO 2 in 40 day old stressed and non-stressed plants of Eleusine coracana. The rate of efflux of 14 C-photosynthates was twice as much in non-stressed plants compared to stressed plants. Young developing leaves, stem apex and stem which are the potential sinks under non-stressed conditions received very little activity under stress conditions. Percent activity in the roots was enhanced under stress suggesting the pattern of translocation was altered under stress conditions. In the plants subjected to moisture stress, after feeding with 14 CO 2 the rate of efflux of 14 C-photosynthates from the fed leaf decreased and the pattern of translocation was altered. Though the effect of stress seems to be directly on the translocation system, the photosynthetic rate appears to be more sensitive to stress than translocation. (author)

  13. Demethoxycurcumin is a potent inhibitor of P-type ATPases from diverse kingdoms of life

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Sehgal, Pankaj; Thanh Tung, Truong

    2016-01-01

    the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site...

  14. Characterization of Na+K+-ATPase in bovine sperm.

    Science.gov (United States)

    Hickey, Katie D; Buhr, Mary M

    2012-04-15

    Existing as a ubiquitous transmembrane protein, Na(+)K(+)-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na(+)K(+)-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na(+)K(+)-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na(+)K(+)-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na(+)K(+)-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Translocation heterozygosity in southern African species of Viscum

    Directory of Open Access Journals (Sweden)

    D. Wiens

    1980-11-01

    Full Text Available Sex-associated and floating translocation complexes are characteristic of dioecious species of  Viscum,  but are virtually absent in monoecious species. The majority of dioecious species has fixed sex-associated translocation complexes with the male being the heterozygous sex. The sex-associated multivalent is usually O4 (ring-of-four or O6 , rarely O8 . Dioecious species without sex-associated translocations are much less common. Most of the dioecious species are also polymorphic for floating translocations, producing one or more additional multivalents ranging from O4 to O12. Floating translocations may be more frequent in species that do not have sex-associated translocations. Supernumerary chromosomes are also present in several species. Sex ratios are at unity in most dioecious species, but female-biased ratios may occur in some species. The high correlation between dioecy and translocation heterozygosity suggests that translocations are primarily associated with the origin and establishment of dioecy. Any róle in the maintenance of biased sex ratios through meiotic drive is probably secondary. Sex-associated translocations may serve to stabilize dioecy by bringing the sex factors into close linkage. Subsequent structural rearrangements within a sex-associated translocation complex may bring the sex factors together in one chromosome pair, releasing floating translocations. The high frequencies of floating translocation heterozygosity in some species indicate that such heterozygosity also has adaptive value.

  16. Annealing enhancement effect by light illumination on proton irradiated Cu(In, Ga)Se2 thin-film solar cells

    International Nuclear Information System (INIS)

    Kawakita, Shirou; Imaizumi, Mitsuru; Matsuda, Sumio; Yamaguchi, Masafumi; Kushiya, Katsumi; Ohshima, Takeshi; Itoh, Hisayoshi

    2002-01-01

    In this paper, we investigated the high radiation tolerance of copper indium gallium di-selenide (CIGS) thin-film solar cells by conducting in situ measurements of short circuit current and open circuit voltage of CIGS thin-film solar cells during and after proton irradiation under short circuit condition. We found that the annealing rate of proton-induced defects in CIGS thin-film solar cells under light illumination with an AM0 solar simulator is higher than that under dark conditions. The activation energy of proton-induced defects in the CIGS thin-film solar cells with (without) light illumination is 0.80 eV (0.92 eV), which implies on enhanced defect annealing rate in CIGS thin-film solar cells due to minority-carrier injection. (author)

  17. Minimizing the cost of translocation failure with decision-tree models that predict species' behavioral response in translocation sites.

    Science.gov (United States)

    Ebrahimi, Mehregan; Ebrahimie, Esmaeil; Bull, C Michael

    2015-08-01

    The high number of failures is one reason why translocation is often not recommended. Considering how behavior changes during translocations may improve translocation success. To derive decision-tree models for species' translocation, we used data on the short-term responses of an endangered Australian skink in 5 simulated translocations with different release conditions. We used 4 different decision-tree algorithms (decision tree, decision-tree parallel, decision stump, and random forest) with 4 different criteria (gain ratio, information gain, gini index, and accuracy) to investigate how environmental and behavioral parameters may affect the success of a translocation. We assumed behavioral changes that increased dispersal away from a release site would reduce translocation success. The trees became more complex when we included all behavioral parameters as attributes, but these trees yielded more detailed information about why and how dispersal occurred. According to these complex trees, there were positive associations between some behavioral parameters, such as fight and dispersal, that showed there was a higher chance, for example, of dispersal among lizards that fought than among those that did not fight. Decision trees based on parameters related to release conditions were easier to understand and could be used by managers to make translocation decisions under different circumstances. © 2015 Society for Conservation Biology.

  18. Tuning of Nafion® by HKUST-1 as coordination network to enhance proton conductivity for fuel cell applications

    International Nuclear Information System (INIS)

    Kim, Hee Jin; Talukdar, Krishan; Choi, Sang-June

    2016-01-01

    Metal-organic frameworks can be intentionally coordinated to achieve improved proton conductivity because they have highly ordered structures and modular nature that serve as a scaffold to anchor acidic groups and develop efficient proton transfer pathways for fuel cell application. Using the concept of a coordination network, the conductivity of Nafion ® was tuned by the incorporation of HKUST-1. It has Cu II –paddle wheel type nodes and 1,3,5-benzenetricarboxylate struts, feature accessible sites that provides an improved protonic channel depending on the water content. In spite of the fact that HKUST-1 is neutral, coordinated water molecules are contributed adequately acidic by Cu II to supply protons to enhance proton conductivity. Water molecules play a vital part in transfer of proton as conducting media and serve as triggers to change proton conductivity through reforming hydrogen bonding networks by water adsorption/desorption process. Increased ion exchange capacity and proton conductivity with lower water uptake of the H 3 PO 4 -doped material, and improved thermal stability (as confirmed by thermogravimetric analysis) were achieved. The structure of HKUST-1 was confirmed via field emission scanning electron microscopy and X-ray diffraction, while the porosity and adsorption desorption capacity were characterized by porosity analysis. Graphical abstract: The H 3 PO 4 -doped HKUST-1/Nafion® composite membrane is demonstrated to be a promising material based on its proton conductivity. HKUST-1 has an average particle diameter of around 15–20 µm. The proton conductivity, IEC values, and the thermal stability of the 2.5 wt% HKUST-1/Nafion® composite membrane suggest that HKUST-1 may be a promising candidate as a proton-conductive material in the polymer electrolyte fuel cell membrane due to its reasonable proton passageway, favorable surface area, lower water uptake with the higher IEC, and proton conductivity of the H 3 PO 4 -doped material and

  19. The proton-pump inhibitor lansoprazole enhances amyloid beta production.

    Science.gov (United States)

    Badiola, Nahuai; Alcalde, Victor; Pujol, Albert; Münter, Lisa-Marie; Multhaup, Gerd; Lleó, Alberto; Coma, Mireia; Soler-López, Montserrat; Aloy, Patrick

    2013-01-01

    A key event in the pathogenesis of Alzheimer's disease (AD) is the accumulation of amyloid-β (Aβ) species in the brain, derived from the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Based on a systems biology study to repurpose drugs for AD, we explore the effect of lansoprazole, and other proton-pump inhibitors (PPIs), on Aβ production in AD cellular and animal models. We found that lansoprazole enhances Aβ37, Aβ40 and Aβ42 production and lowers Aβ38 levels on amyloid cell models. Interestingly, acute lansoprazole treatment in wild type and AD transgenic mice promoted higher Aβ40 levels in brain, indicating that lansoprazole may also exacerbate Aβ production in vivo. Overall, our data presents for the first time that PPIs can affect amyloid metabolism, both in vitro and in vivo.

  20. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

    Directory of Open Access Journals (Sweden)

    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  1. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    Science.gov (United States)

    Nagao, Yuki; Kubo, Takahiro

    2014-12-01

    Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120-670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system.

  2. The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host.

    Science.gov (United States)

    Dzioba-Winogrodzki, Judith; Winogrodzki, Olga; Krulwich, Terry A; Boin, Markus A; Häse, Claudia C; Dibrov, Pavel

    2009-01-01

    The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li(+), Na(+), and K(+) ions in pH-dependent manner with maximal activity at pH 9.0-9.5. Exchange was electrogenic (more than one H(+) translocated per cation moved in opposite direction). The apparent K(m) at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li(+), Na(+), and K(+), respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems. Copyright 2008 S. Karger AG, Basel.

  3. Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

    Science.gov (United States)

    Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

    2011-01-01

    Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

  4. Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase*

    Science.gov (United States)

    Morton, Michael J.; Farr, Glen A.; Hull, Michael; Capendeguy, Oihana; Horisberger, Jean-Daniel; Caplan, Michael J.

    2010-01-01

    Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys54 in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys54 α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit. PMID:20801885

  5. Na+/K+-ATPase: Activity and inhibition

    Science.gov (United States)

    Čolović, M.; Krstić, D.; Krinulović, K.; Momić, T.; Savić, J.; Vujačić, A.; Vasić, V.

    2009-09-01

    The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.

  6. Excited state proton transfer in strongly enhanced GFP (sGFP2).

    Science.gov (United States)

    van Oort, Bart; ter Veer, Mirelle J T; Groot, Marie Louise; van Stokkum, Ivo H M

    2012-07-07

    Proton transfer is an elementary process in biology. Green fluorescent protein (GFP) has served as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. We have used pump-dump-probe spectroscopy to study how proton transfer through the 'proton-wire' around the chromophore is affected by a combination of mutations in a modern GFP variety (sGFP2). The results indicate that in H(2)O, after absorption of a photon, a proton is transferred (A* → I*) in 5 ps, and back-transferred from a ground state intermediate (I → A) in 0.3 ns, similar to time constants found with GFPuv, although sGFP2 shows less heterogeneous proton transfer. This suggests that the mutations left the proton-transfer largely unchanged, indicating the robustness of the proton-wire. We used pump-dump-probe spectroscopy in combination with target analysis to probe suitability of the sGFP2 fluorophore for super-resolution microscopy.

  7. Contrast-Enhanced Proton Radiography for Patient Set-up by Using X-Ray CT Prior Knowledge

    Energy Technology Data Exchange (ETDEWEB)

    Spadea, Maria Francesca, E-mail: mfspadea@unicz.it [Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro (Italy); Fassi, Aurora [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano (Italy); Zaffino, Paolo [Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro (Italy); Riboldi, Marco; Baroni, Guido [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano (Italy); Bioengineering Unit—CNAO Foundation, Pavia (Italy); Depauw, Nicolas [Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts (United States); Centre for Medical Radiation Physics, University of Wollongong, Wollongong (Australia); Seco, Joao [Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts (United States)

    2014-11-01

    Purpose: To obtain a contrasted image of the tumor region during the setup for proton therapy in lung patients, by using proton radiography and x-ray computed tomography (CT) prior knowledge. Methods and Materials: Six lung cancer patients' CT scans were preprocessed by masking out the gross tumor volume (GTV), and digitally reconstructed radiographs along the planned beam's eye view (BEV) were generated, for a total of 27 projections. Proton radiographies (PR) were also computed for the same BEV through Monte Carlo simulations. The digitally reconstructed radiograph was subtracted from the corresponding proton image, resulting in a contrast-enhanced proton radiography (CEPR). Michelson contrast analysis was performed both on PR and CEPR. The tumor region was then automatically segmented on CEPR and compared to the ground truth (GT) provided by physicians in terms of Dice coefficient, accuracy, precision, sensitivity, and specificity. Results: Contrast on CEPR was, on average, 4 times better than on PR. For 10 lateral projections (±45° off of 90° or 270°), although it was not possible to distinguish the tumor region in the PR, CEPR offers excellent GTV visibility. The median ± quartile values of Dice, precision, and accuracy indexes were 0.86 ± 0.03, 0.86 ± 0.06, and 0.88 ± 0.02, respectively, thus confirming the reliability of the method in highlighting tumor boundaries. Sensitivity and specificity analysis demonstrated that there is no systematic over- or underestimation of the tumor region. Identification of the tumor boundaries using CEPR resulted in a more accurate and precise definition of GTV compared to that obtained from pretreatment CT. Conclusions: In most proton centers, the current clinical protocol is to align the patient using kV imaging with bony anatomy as a reference. We demonstrated that CEPR can significantly improve tumor visualization, allowing better patient set-up and permitting image guided proton therapy (IGPT)

  8. Abdominal radiation causes bacterial translocation

    International Nuclear Information System (INIS)

    Guzman-Stein, G.; Bonsack, M.; Liberty, J.; Delaney, J.P.

    1989-01-01

    The purpose of this study was to determine if a single dose of radiation to the rat abdomen leads to bacterial translocation into the mesenteric lymph nodes (MLN). A second issue addressed was whether translocation correlates with anatomic damage to the mucosa. The radiated group (1100 cGy) which received anesthesia also was compared with a control group and a third group which received anesthesia alone but no abdominal radiation. Abdominal radiation lead to 100% positive cultures of MLN between 12 hr and 4 days postradiation. Bacterial translocation was almost nonexistent in the control and anesthesia group. Signs of inflammation and ulceration of the intestinal mucosa were not seen until Day 3 postradiation. Mucosal damage was maximal by Day 4. Bacterial translocation onto the MLN after a single dose of abdominal radiation was not apparently dependent on anatomical, histologic damage of the mucosa

  9. The AAA protein spastin possesses two levels of basal ATPase activity.

    Science.gov (United States)

    Fan, Xiangyu; Lin, Zhijie; Fan, Guanghui; Lu, Jing; Hou, Yongfei; Habai, Gulijiazi; Sun, Linyue; Yu, Pengpeng; Shen, Yuequan; Wen, Maorong; Wang, Chunguang

    2018-04-30

    The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected. © 2018 Federation of European Biochemical Societies.

  10. Enhancement in Proton Conductivity and Thermal Stability in Nafion Membranes Induced by Incorporation of Sulfonated Carbon Nanotubes.

    Science.gov (United States)

    Yin, Chongshan; Li, Jingjing; Zhou, Yawei; Zhang, Haining; Fang, Pengfei; He, Chunqing

    2018-04-25

    Proton exchange membrane fuel cell (PEMFC) is one of the most promising green power sources, in which perfluorinated sulfonic acid ionomer-based membranes (e.g., Nafion) are widely used. However, the widespread application of PEMFCs is greatly limited by the sharp degradation in electrochemical properties of the proton exchange membranes under high temperature and low humidity conditions. In this work, the high-performance sulfonated carbon nanotubes/Nafion composite membranes (Su-CNTs/Nafion) for the PEMFCs were prepared and the mechanism of the microstructures on the macroscopic properties of membranes was intensively studied. Microstructure evolution in Nafion membranes during water uptake was investigated by positron annihilation lifetime spectroscopy, and results strongly showed that the Su-CNTs or CNTs in Nafion composite membranes significantly reinforced Nafion matrices, which influenced the development of ionic-water clusters in them. Proton conductivities in Su-CNTs/Nafion composite membranes were remarkably enhanced due to the mass formation of proton-conducting pathways (water channels) along the Su-CNTs. In particular, these pathways along Su-CNTs in Su-CNTs/Nafion membranes interconnected the isolated ionic-water clusters at low humidity and resulted in less tortuosity of the water channel network for proton transportation at high humidity. At a high temperature of 135 °C, Su-CNTs/Nafion membranes maintained high proton conductivity because the reinforcement of Su-CNTs on Nafion matrices reduced the evaporation of water molecules from membranes as well as the hydrophilic Su-CNTs were helpful for binding water molecules.

  11. Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

    Science.gov (United States)

    Rolloos, Martijn; Hooykaas, Paul J J; van der Zaal, Bert J

    2015-02-09

    Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

  12. Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages.

    Science.gov (United States)

    Schachter, Julieta; Delgado, Kelly Valcárcel; Barreto-de-Souza, Victor; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis; Meyer-Fernandes, José Roberto

    2015-05-01

    Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages. Copyright © 2014 Elsevier GmbH. All rights reserved.

  13. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

    Directory of Open Access Journals (Sweden)

    Tohru Minamino

    2016-03-01

    Full Text Available The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

  14. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

    Science.gov (United States)

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

  15. Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

    Science.gov (United States)

    Gatto, C; Lutsenko, S; Shin, J M; Sachs, G; Kaplan, J H

    1999-05-14

    The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

  16. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, Yuki, E-mail: ynagao@jaist.ac.jp; Kubo, Takahiro

    2014-12-30

    Graphical abstract: - Highlights: • Proton transport of fully protonated poly(aspartic acid) thin film was investigated. • The thin film structure differed greatly from the partially protonated one. • Proton transport occurs on the surface, not inside of the thin film. • This result contributes to biological transport systems such as bacteriorhodopsin. - Abstract: Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120–670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system.

  17. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    International Nuclear Information System (INIS)

    Nagao, Yuki; Kubo, Takahiro

    2014-01-01

    Graphical abstract: - Highlights: • Proton transport of fully protonated poly(aspartic acid) thin film was investigated. • The thin film structure differed greatly from the partially protonated one. • Proton transport occurs on the surface, not inside of the thin film. • This result contributes to biological transport systems such as bacteriorhodopsin. - Abstract: Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120–670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system

  18. Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells

    Directory of Open Access Journals (Sweden)

    Mitsushi J. Ikemoto

    2017-09-01

    Full Text Available Addicsin (Arl6ip5 is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1 and participates in the regulation of intracellular glutathione (GSH content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ, a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABAA receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na+/K+ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABAA receptor antagonist, picrotoxin, but not by a competitive GABAA receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.

  19. Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

    Science.gov (United States)

    Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

    1997-03-15

    The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes

  20. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor.

    Science.gov (United States)

    Mao, Huzhang; Saha, Mitul; Reyes-Aldrete, Emilio; Sherman, Michael B; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J; Morais, Marc C

    2016-03-01

    Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Determination of dose enhancement caused by gold-nanoparticles irradiated with proton, X-rays (kV and MV) and electron beams, using alanine/EPR dosimeters

    International Nuclear Information System (INIS)

    Smith, Clare L.; Ackerly, Trevor; Best, Stephen P.; Gagliardi, Frank; Kie, Katahira; Little, Peter J.; McCorkell, Giulia; Sale, Charlotte A.; Tsunei, Yusuke; Tominaga, Takahiro; Volaric, Sioe See; Geso, Moshi

    2015-01-01

    The main aims of this research was to employ alanine doped with gold-nanoparticles “AuNPs” to determine the levels of dose enhancement caused by these particles when irradiated with proton beams, low and high energy X-rays and electrons. DL-alanine was impregnated with 5 nm gold-nanoparticles (3% by weight) and added as a uniform layer within a wax pellet of dimensions 10 × 5 × 5 mm. Control pellets, containing DL-Alanine were also produced, and placed within a phantom, and exposed to various types of radiations: low energy (kV ranges) X-rays were obtained from a superficial machine, high energy (MV) X-rays and electrons derived from a linear accelerator, and protons were produced by the Hyogo Ion Beam Centre in Japan. Nominal doses received ranged from 2 to 20 Gy (within clinical range). The Electron Paramagnetic Resonance (EPR) spectra of the irradiated samples were recorded on a BRUKER Elexsys 9.5 MHz. The dose enhancement caused by gold nanoparticles for 80 kV x-rays was found to be more than 60% at about 5 Gy. Smaller dose enhancements (under the same measurement conditions) were observed for megavoltage x-ray beams (up to 10%). Dose enhancement caused by charged particles indicated minimal values for 6 MeV electrons (approximately 5%) whilst less than that is obtained with protons of 150 MeV. The proton results validate the latest simulation results based on Monte Carlo calculations but the dose enhancement is significantly less than that reported in cell and animal model systems, (about 20%). We attribute this difference to the fact that alanine only measures the levels of free radicals generated by the inclusion of nanoparticles and not the redox type radicals (such as reactive oxygen species) generated from aqueous media in cells. Dose enhancement caused by 5 nm gold-nanoparticles with radiotherapy type proton beams has been found to be less than 5% as determined when using alanine/wax as both a phantom and dosimeter. This agrees well

  2. The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis.

    Science.gov (United States)

    Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J

    2008-09-19

    Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

  3. Reciprocal translocations in mice taken from the thirty-kilometer zone around the crippled reactor of Chernobyl

    International Nuclear Information System (INIS)

    Pomerantseva, M.D.; Ramajya, L.K.; Testov, B.V.; Chekhovich, A.V.; Shevchenko, V.A.; Shaks, A.I.; Lobaneva, N.V.

    1990-01-01

    A study was made of the incidence of genetic damages to germ cells of male mice taken from or exposed within the thirty-kilometer Zone of Chernobyl, the contaminated no-man's-land around the reactor that failed. At all contamination levels mouse spermatocytes exhibited reciprocal translocations, a relatively low frequency of which increased with increasing dose rate. Heterozygotes, with respect to reciprocal translocations (5%), were found among males exposed to enhanced radiation background as early embryons

  4. Insulin stimulation of [3H]-ouabain binding to cerebrovascular (Na+ + K+)-ATPase

    International Nuclear Information System (INIS)

    Caspers, M.L.; Grammas, P.

    1986-01-01

    Brain microvessels were isolated from rat cerebral cortices. The binding of [ 3 H]-ouabain to microvascular (Na + + K + )-ATPase increased with microvessel protein (37-110μg) and was time dependent with maximum binding observed at 15 min at 37 0 C. Non-specific binding, measured in the presence of 50μM ouabain, was less than 2% of total binding. Scatchard analysis of preliminary [ 3 H]-ouabain binding data yielded a K/sub D/ of 44nM and a B/sub max/ of 12pmol/mg. Since the high affinity (α+) form of the enzyme is purportedly hormonally regulated, the effect of insulin on [ 3 H]-ouabain binding to microvessels was studied. Insulin (0.001-10μM) stimulation of [ 3 H]-ouabain binding was dose dependent. To assess whether this was a specific or a peptide-protective effect, assays were performed in the presence of bovine serum albumin (BSA). Addition of BSA (10μM) enhanced the amount of [ 3 H]-ouabain bound 4-fold. Further increases in the BSA concentration (20μM) did not increase binding. Addition of 10μM insulin evoked a 20% increase in [ 3 H]-ouabain binding above BSA-treated controls. In summary, the data suggest that the (α+) form of the (Na + + K + )-ATPase is present in cerebral endothelium and [ 3 H]-ouabain binding is significantly elevated by insulin in a dose dependent manner. Therefore, insulin may regulate microvascular (Na + + K + )-ATPase and thus be a modulator of blood-brain permeability to ions

  5. Carbonylation Modification Regulates Na/K-ATPase Signaling and Salt Sensitivity: A Review and a Hypothesis.

    Science.gov (United States)

    Shah, Preeya T; Martin, Rebecca; Yan, Yanling; Shapiro, Joseph I; Liu, Jiang

    2016-01-01

    Na/K-ATPase signaling has been implicated in different physiological and pathophysiological conditions. Accumulating evidence indicates that oxidative stress not only regulates the Na/K-ATPase enzymatic activity, but also regulates its signaling and other functions. While cardiotonic steroids (CTS)-induced increase in reactive oxygen species (ROS) generation is an intermediate step in CTS-mediated Na/K-ATPase signaling, increase in ROS alone also stimulates Na/K-ATPase signaling. Based on literature and our observations, we hypothesize that ROS have biphasic effects on Na/K-ATPase signaling, transcellular sodium transport, and urinary sodium excretion. Oxidative modulation, in particular site specific carbonylation of the Na/K-ATPase α1 subunit, is a critical step in proximal tubular Na/K-ATPase signaling and decreased transcellular sodium transport leading to increases in urinary sodium excretion. However, once this system is overstimulated, the signaling, and associated changes in sodium excretion are blunted. This review aims to evaluate ROS-mediated carbonylation of the Na/K-ATPase, and its potential role in the regulation of pump signaling and sodium reabsorption in the renal proximal tubule (RPT).

  6. Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

    International Nuclear Information System (INIS)

    Pougeois, R.; Lauquin, G.J.

    1985-01-01

    The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (P /sub i/ ), could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca 2+ -ATPase in the presence of Ca 2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase, abolished P /sub i/ binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites

  7. The modulation of erythrocyte Na+/K+-ATPase activity by curcumin

    Directory of Open Access Journals (Sweden)

    Prabhakar Singh

    2015-11-01

    Full Text Available Curcumin, an active biphenolic molecule present in turmeric (Curcuma longa, has been reported to elicit plethora of health protective effects. The present study was carried out in vitro, in vivo and in silico to investigate the modulatory effects of curcumin on erythrocyte membrane Na+/K+-ATPase activity. In vitro curcumin (10−5 M to 10−8 M was incubated with human erythrocytes membrane. In vivo curcumin (340 mg/kg b.w. and 170 mg/kg b.w. was supplemented to wistar rats for 21 days. In silico, catalytic unit α of Na+/K+-ATPase (3b8e.pdb protein was used as a receptor for the natural ligand ATP to study curcumin-mediated docking simulation using AutoDock4. The in vitro effect of curcumin on the Na+/K+-ATPase activity in human erythrocytes was biphasic. An inhibitory response was observed at 10−5 M (p < 0.001. An activation of the Na+/K+-ATPase activity was observed at 10−7 and 10−8 M (p < 0.001 and p < 0.01. In vivo, curcumin supplementation to rats increased the Na+/K+-ATPase activity at doses 340 mg/kg b.w. (p < 0.001 as well as at 170 mg/kg b.w., (p < 0.01. AutoDock4 docking simulation study showed that both ligands curcumin and ATP actively interacted with amino acids Glu214, Ser215, Glu216, Thr371, Asn377, Arg378, Met379, Arg438, Val440, Ala444, Lys451 and Asp586 at the catalytic cavity of Na+/K+-ATPase. ATP had more H bonding and hydrophobic interaction with active site amino acid residues compared to curcumin. These finding may explain some of the health beneficial properties of curcumin associated with deregulated Na+/K+-ATPase activity or ions homeostasis.

  8. Na/K Pump and Beyond: Na/K-ATPase as a Modulator of Apoptosis and Autophagy

    Directory of Open Access Journals (Sweden)

    Cassiano Felippe Gonçalves-de-Albuquerque

    2017-04-01

    Full Text Available Lung cancer is a leading cause of global cancer deaths. Na/K-ATPase has been studied as a target for cancer treatment. Cardiotonic steroids (CS trigger intracellular signalling upon binding to Na/K-ATPase. Normal lung and tumour cells frequently express different pump isoforms. Thus, Na/K-ATPase is a powerful target for lung cancer treatment. Drugs targeting Na/K-ATPase may induce apoptosis and autophagy in transformed cells. We argue that Na/K-ATPase has a role as a potential target in chemotherapy in lung cancer treatment. We discuss the effects of Na/K-ATPase ligands and molecular pathways inducing deleterious effects on lung cancer cells, especially those leading to apoptosis and autophagy.

  9. Na/K Pump and Beyond: Na/K-ATPase as a Modulator of Apoptosis and Autophagy.

    Science.gov (United States)

    Felippe Gonçalves-de-Albuquerque, Cassiano; Ribeiro Silva, Adriana; Ignácio da Silva, Camila; Caire Castro-Faria-Neto, Hugo; Burth, Patrícia

    2017-04-21

    Lung cancer is a leading cause of global cancer deaths. Na/K-ATPase has been studied as a target for cancer treatment. Cardiotonic steroids (CS) trigger intracellular signalling upon binding to Na/K-ATPase. Normal lung and tumour cells frequently express different pump isoforms. Thus, Na/K-ATPase is a powerful target for lung cancer treatment. Drugs targeting Na/K-ATPase may induce apoptosis and autophagy in transformed cells. We argue that Na/K-ATPase has a role as a potential target in chemotherapy in lung cancer treatment. We discuss the effects of Na/K-ATPase ligands and molecular pathways inducing deleterious effects on lung cancer cells, especially those leading to apoptosis and autophagy.

  10. Human and rodent muscle Na(+)-K(+)-ATPase in diabetes related to insulin, starvation, and training

    DEFF Research Database (Denmark)

    Schmidt, T A; Hasselbalch, S; Farrell, P A

    1994-01-01

    cerebral cortex Na(+)-K(+)-ATPase concentration as a result of diabetes, semistarvation, or insulin treatment. In human subjects, Na(+)-K(+)-ATPase concentration in vastus lateralis muscle biopsies was 17 and 22% greater (P dependent diabetes...... mellitus (n = 24) and insulin-dependent diabetes mellitus (n = 7) than in control subjects (n = 8). A positive linear correlation between muscle Na(+)-K(+)-ATPase and plasma insulin concentrations was observed (r = 0.50, P = 0.006; n = 29). Thus, insulin seems a regulator of muscle Na......(+)-K(+)-ATPase concentration, reduction of muscle Na(+)-K(+)-ATPase concentration with untreated diabetes bears similarities with undernourishment, and physical conditioning may ameliorate the muscle Na(+)-K(+)-ATPase concentration decrease induced by diabetes....

  11. Tuning of Nafion{sup ®} by HKUST-1 as coordination network to enhance proton conductivity for fuel cell applications

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hee Jin, E-mail: zammanbo814@knu.ac.kr [Kyungpook National University, Research Institute of Advanced Energy Technology (Korea, Republic of); Talukdar, Krishan, E-mail: krishantu@yahoo.com; Choi, Sang-June, E-mail: sjchoi@knu.ac.kr [Kyungpook National University, Department of Environmental Engineering (Korea, Republic of)

    2016-02-15

    Metal-organic frameworks can be intentionally coordinated to achieve improved proton conductivity because they have highly ordered structures and modular nature that serve as a scaffold to anchor acidic groups and develop efficient proton transfer pathways for fuel cell application. Using the concept of a coordination network, the conductivity of Nafion{sup ®} was tuned by the incorporation of HKUST-1. It has Cu{sup II}–paddle wheel type nodes and 1,3,5-benzenetricarboxylate struts, feature accessible sites that provides an improved protonic channel depending on the water content. In spite of the fact that HKUST-1 is neutral, coordinated water molecules are contributed adequately acidic by Cu{sup II} to supply protons to enhance proton conductivity. Water molecules play a vital part in transfer of proton as conducting media and serve as triggers to change proton conductivity through reforming hydrogen bonding networks by water adsorption/desorption process. Increased ion exchange capacity and proton conductivity with lower water uptake of the H{sub 3}PO{sub 4}-doped material, and improved thermal stability (as confirmed by thermogravimetric analysis) were achieved. The structure of HKUST-1 was confirmed via field emission scanning electron microscopy and X-ray diffraction, while the porosity and adsorption desorption capacity were characterized by porosity analysis. Graphical abstract: The H{sub 3}PO{sub 4}-doped HKUST-1/Nafion® composite membrane is demonstrated to be a promising material based on its proton conductivity. HKUST-1 has an average particle diameter of around 15–20 µm. The proton conductivity, IEC values, and the thermal stability of the 2.5 wt% HKUST-1/Nafion® composite membrane suggest that HKUST-1 may be a promising candidate as a proton-conductive material in the polymer electrolyte fuel cell membrane due to its reasonable proton passageway, favorable surface area, lower water uptake with the higher IEC, and proton conductivity of the H

  12. Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

    Directory of Open Access Journals (Sweden)

    Qi Zhou

    2016-05-01

    Full Text Available Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM, GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor and U73122 (PLC inhibitor. However, 2-APB (IP3R blocker only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

  13. Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells

    International Nuclear Information System (INIS)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J.

    1988-01-01

    Dopamine, generated locally from L-dopa, inhibits Na + -K + -ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na + -K + -ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (Qo 2 ) and 86 Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive Qo 2 or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive Qo 2 in a concentration-dependent manner, with half-maximal inhibition (K 0.5 ) of 5 x 10 -7 M and a maximal inhibition of 14.1 ± 1.5% at 10 -4 M. L-Dopa also blunted the nystatin-stimulated Qo 2 in a concentration-dependent manner, indicating the L-dopa directly inhibits Na + -K + -ATPase activity and not sodium entry. Ouabain-sensitive 86 Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive Qo 2 and 86 Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive Qo 2 at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na + -K + -ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner

  14. General and specific lipid-protein interactions in Na,K-ATPase.

    Science.gov (United States)

    Cornelius, F; Habeck, M; Kanai, R; Toyoshima, C; Karlish, S J D

    2015-09-01

    The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions." Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Na,K-ATPase structure/function relationships probed by the denaturant urea.

    Science.gov (United States)

    Esmann, Mikael; Fedosova, Natalya U; Olesen, Claus

    2015-05-01

    Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37°C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K+ as well as the K+-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na+. Half-maximal inhibition is seen with about 1M urea for shark enzyme and about 2M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

    NARCIS (Netherlands)

    Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

  17. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

    Science.gov (United States)

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-05-16

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

  18. Copper-transporting P-type ATPases use a unique ion-release pathway

    DEFF Research Database (Denmark)

    Andersson, Magnus; Mattle, Daniel; Sitsel, Oleg

    2014-01-01

    Heavy metals in cells are typically regulated by PIB-type ATPases. The first structure of the class, a Cu(+)-ATPase from Legionella pneumophila (LpCopA), outlined a copper transport pathway across the membrane, which was inferred to be occluded. Here we show by molecular dynamics simulations...... that extracellular water solvated the transmembrane (TM) domain, results indicative of a Cu(+)-release pathway. Furthermore, a new LpCopA crystal structure determined at 2.8-Å resolution, trapped in the preceding E2P state, delineated the same passage, and site-directed-mutagenesis activity assays support...... a functional role for the conduit. The structural similarities between the TM domains of the two conformations suggest that Cu(+)-ATPases couple dephosphorylation and ion extrusion differently than do the well-characterized PII-type ATPases. The ion pathway explains why certain Menkes' and Wilson's disease...

  19. The effects of dexamethasone on the Na,K-ATPase activity and pump function of corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo

    2009-05-01

    The Na(+)- and K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase alpha(1)-subunit. Dexamethasone (1 or 10 microM) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase alpha(1)-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase alpha(1)-subunit. Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase alpha(1)-subunits.

  20. Partial purification of the ATP-driven calcium pump of Streptococcus sanguis

    International Nuclear Information System (INIS)

    Lynn, A.R.; Rosen, B.P.

    1986-01-01

    ATP-dependent transport of calcium has been observed in several species of streptococci as uptake of 45 Ca 2+ into everted membrane vesicles. Membranes from Streptococcus sanguis and Streptococcus faecalis were solubilized with octyl-β-D-glucoside or Triton X-100, and the extracts reconstituted into proteoliposomes containing Escherichia coli or soybean phospholipid. Calcium transport in reconstituted proteoliposomes was insensitive to the ionophores nigericin and valinomycin and was unaffected by the F 0 F 1 inhibitor N,N'-dicyclohexylcarbodiimide. Uptake was inhibited by ortho-vanadate with a K/sub i/ in the micromolar range. These results demonstrate that the reconstituted transport activities are not the result of ATP-driven proton pumping via the F 0 F 1 coupled to a calcium/proton antiporter and suggest that existence of a calcium translocating ATPase. Partial purification of the transport activity from Streptococcus sanguis has been achieved using density gradient centrifugation and FPLC

  1. The effect of O-GlcNAcylation on hnRNP A1 translocation and interaction with transportin1

    Energy Technology Data Exchange (ETDEWEB)

    Roth, Shira; Khalaila, Isam, E-mail: isam@bgu.ac.il

    2017-01-01

    The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a major pre-mRNA binding protein involved in transcription and translation. Although predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytosol, delivering its anchored pre-mRNA for further processing. Translocation is important for hnRNP A1 to accomplish its transcriptional and translational roles. Transportin1 (Trn1), a translocation protein, facilitates the translocation of hnRNP A1 back to the nucleus. Moreover, phosphorylation of serine residues at hnRNP A1 C-terminal domain affects its translocation. In this study, we found that phosphorylation is not the only modification that hnRNP A1 undergoes, but also O-linked N-acetylglucosaminylation (O-GlcNAcylation) could occur. Several putative novel O-GlcNAcylation and phosphorylation sites in hnRNP A1 were mapped. Whereas enhanced O-GlcNAcylation increased hnRNP A1 interaction with Trn1, enhanced phosphorylation reduced the interaction between the proteins. In addition, elevated O-GlcNAcylation resulted in hnRNP A1 seclusion in the nucleus, whereas elevated phosphorylation resulted in its accumulation in the cytosol. These findings suggest that a new player, i.e., O-GlcNAcylation, regulates hnRNP A1 translocation and interaction with Trn1, possibly affecting its function. There is a need for further study, to elucidate the role of O-GlcNAcylation in the regulation of the specific activities of hnRNP A1 in transcription and translation. - Highlights: • O-GlcNAcylation regulates hnRNP A1 translocation and interaction with Trn1. • Reciprocity between phosphorylation and O-GlcNAcylation in hnRNP A1 is proposed. • Novel O-GlcNAcylation and phosphorylation sites on hnRNPA1 were identified.

  2. The influence of blood plasma of irradiated animals on activity of Ca2+ - ATPase and Mg2+ - ATPase in plasma membrane of thymocytes

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1994-01-01

    Rats were irradiated at doses 1.5, 4.0, 7.0 and 10 Gy. After 1, 8, 15, 22 and 30 days the effect of blood plasma on activity of Ca 2+ -ATPase and Mg 2+ -ATPase in plasma membrane of thymocytes was investigated. It was found that the raise of irradiation dose leads to increasing of blood plasma effect on membrane-bound enzymes

  3. Enhanced proton conductivity of niobium phosphates by interfacing crystal grains with an amorphous functional phase

    DEFF Research Database (Denmark)

    Huang, Yunjie; Yu, Lele; Li, Haiyan

    2016-01-01

    Niobium phosphate is an interesting proton conductor operational in the intermediate temperature range. In the present work two forms of phosphates were prepared: an amorphous one with high specific area and a crystalline one with low specific surface area. Both phosphates exhibited very low prot...... the high surface area amorphous phosphate was used as the precursor. At 250 °C thus obtained niobium phosphate showed a high and stable conductivity of 0.03 S cm−1 under dry atmosphere and of 0.06 S cm−1 at a water partial pressure of 0.12 atm....... conductivities. An activation process was developed to convert the phosphates into crystal grains with a phosphorus rich amorphous phase along the grain boundaries. As a result, the obtained niobium phosphates showed considerably enhanced and stable proton conductivities. The activation effect was prominent when...

  4. Protein import into chloroplasts requires a chloroplast ATPase

    International Nuclear Information System (INIS)

    Pain, D.; Blobel, G.

    1987-01-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [ 35 S]methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H + , K + , Na + , or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors

  5. Protein import into chloroplasts requires a chloroplast ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pain, D.; Blobel, G.

    1987-05-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

  6. Structural and functional studies of heavy metal ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg

    2015-01-01

    to handle heavy metal ions. LpCopA is then compared to its two human homologues ATP7A and ATP7B, which cause the severe Menkes and Wilson diseases when malfunctioning. The differences between the three proteins are described and disease-causing mutations in the human proteins are analyzed. The crystal......Copper and zinc are trace elements that are crucial for the well-being of all cells and are an indispensable part of many proteins. At the same time, the intracellular levels of these metals require careful regulation, as an excess or deficiency may be lethal. P1B-ATPases are key players in Cu......+ and Zn2+ homeostasis that belong to the superfamily of P-type ATPases, transmembrane proteins which are present in virtually all lifeforms, with functions ranging from membrane potential generation to muscle relaxation. The goal of this thesis is to improve our understanding of P1B-ATPases by focusing...

  7. Financial costs of large carnivore translocations--accounting for conservation.

    Science.gov (United States)

    Weise, Florian J; Stratford, Ken J; van Vuuren, Rudolf J

    2014-01-01

    Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars). Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23), and $2,108 per leopard (n = 6). One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%), followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4%) of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown) with a strong species bias. Four leopards (66.7%) were successfully translocated but only eight of the 20 cheetahs (40.0%) with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC) and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

  8. Financial costs of large carnivore translocations--accounting for conservation.

    Directory of Open Access Journals (Sweden)

    Florian J Weise

    Full Text Available Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars. Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23, and $2,108 per leopard (n = 6. One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%, followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4% of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown with a strong species bias. Four leopards (66.7% were successfully translocated but only eight of the 20 cheetahs (40.0% with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

  9. Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

    Science.gov (United States)

    Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

    1999-07-01

    The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

  10. Radiation induced reciprocal translocations and inversions in anopheles albimanus

    International Nuclear Information System (INIS)

    Kaiser, P.E.; Seawright, J.A.; Benedict, M.Q.; Narang, S.

    1982-01-01

    Reciprocal translocations and inversions were induced in Anopheles albimanus Wiedemann by irradiation of males with X rays. A total of 1669 sperm were assayed, and 175 new aberrations were identified as follows: 102 reciprocal translocations (67 autosomal and 35 sex-linked), 45 pericentric inversions, and 28 paracentric inversions. Eleven of the translocations were nearly whole-arm interchanges, and these were selected for the construction of 'capture systems' for compound chromosomes. Two double-heterozygous translocation strains and four homozygous translocation strains were established. Anopheles albimanus females were irradiated, and a pseudolinkage scheme involving mutant markers was employed to identify reciprocal translocations. The irradiation of females was very inefficient; only one translocation was recovered from 1080 ova tested

  11. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    . The functionality on the P4-ATPase complex is essential for several cellular processes, such as vesicle-mediated transport. However, the specific role of flippase activity in vesicle biogenesis and the regulatory mechanism behind this process is still poorly understood. In these studies, we identified...... affordable alternative using a microscope-based cytometer. This system can simultaneously provide information on flippase activity and expression levels. Taken together, the findings described in this thesis provide new tools for P4-ATPase characterization and valuable insights into the regulation...

  12. Study on the changes in the levels of membrane-bound ATPases ...

    African Journals Online (AJOL)

    An attempt has been made to determine the deleterious effects of λ cyhalothrin- induced in fresh water tilapia (Oreochromis mossambicus) with respect to changes in the activities of membrane-bound ATPases (Na+/K+, Mg+ and Ca2+ ATPase) and mineral status ...

  13. Stereochemistry-Dependent Proton Conduction in Proton Exchange Membrane Fuel Cells.

    Science.gov (United States)

    Thimmappa, Ravikumar; Devendrachari, Mruthyunjayachari Chattanahalli; Kottaichamy, Alagar Raja; Tiwari, Omshanker; Gaikwad, Pramod; Paswan, Bhuneshwar; Thotiyl, Musthafa Ottakam

    2016-01-12

    Graphene oxide (GO) is impermeable to H2 and O2 fuels while permitting H(+) shuttling, making it a potential candidate for proton exchange membrane fuel cells (PEMFC), albeit with a large anisotropy in their proton transport having a dominant in plane (σIP) contribution over the through plane (σTP). If GO-based membranes are ever to succeed in PEMFC, it inevitably should have a dominant through-plane proton shuttling capability (σTP), as it is the direction in which proton gets transported in a real fuel-cell configuration. Here we show that anisotropy in proton conduction in GO-based fuel cell membranes can be brought down by selectively tuning the geometric arrangement of functional groups around the dopant molecules. The results show that cis isomer causes a selective amplification of through-plane proton transport, σTP, pointing to a very strong geometry angle in ionic conduction. Intercalation of cis isomer causes significant expansion of GO (001) planes involved in σTP transport due to their mutual H-bonding interaction and efficient bridging of individual GO planes, bringing down the activation energy required for σTP, suggesting the dominance of a Grotthuss-type mechanism. This isomer-governed amplification of through-plane proton shuttling resulted in the overall boosting of fuel-cell performance, and it underlines that geometrical factors should be given prime consideration while selecting dopant molecules for bringing down the anisotropy in proton conduction and enhancing the fuel-cell performance in GO-based PEMFC.

  14. Relationship between serum adiponectin level and ATPase activity of erythrocyte membrance in patients with 2-type diabetes

    International Nuclear Information System (INIS)

    Song Jiejin

    2008-01-01

    Objective: To explore the possible mechanism of development nephrosis as related to changes of serum adiponectin levels and alteration of activities of Na + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase of erythrocyte membrance in patients with 2-type diabetes. Methods: Serum adiponectin levels (with RIA) and erythrocyte membrance (prepared with Reilnila method) Na + ·K + - ATPase and Ca +2 ·Mg +2 -ATPase activity were determined in 45 DM2 patients without nephropathy, 31 DM2 patients with nephropathy and 30 controls. Results: Serum adiponectin levels in the diabetic patients were significantly lower than those in controls (P + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase activities were also significantly lower than those in controls (P + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase activities of erythrocyte membrance. (authors)

  15. Increasing acidification of nonreplicating Lactococcus lactis Delta thyA mutants by incorporating ATPase activity

    DEFF Research Database (Denmark)

    Pedersen, Martin Bastian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

    2002-01-01

    % of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing...... nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by noureplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We...... concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis....

  16. The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis*

    Science.gov (United States)

    Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J.

    2008-01-01

    Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ± 0.2 × 10-4 s-1) at 25 °C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain. PMID:18632665

  17. Adaptive plasticity of skeletal muscle energetics in hibernating frogs: mitochondrial proton leak during metabolic depression.

    Science.gov (United States)

    Boutilier, Robert G; St-Pierre, Julie

    2002-08-01

    properties of the mitochondria. In the absence of oxygen, the mitochondrial F(1)F(o)-ATPase (the ATP synthase) begins to run backwards as it actively pumps protons from the matrix in an attempt to maintain the mitochondrial membrane potential. At this time, the ATP synthase functions as an ATPase to preserve a certain proton-motive force. Frogs limit ATP wastage during anoxia by a profound inhibition of the ATP synthase. Taken together, our studies show that protonmotive force is lowered aerobically by restricting electron supply and during anoxia by restricting mitochondrial ATPase activity.

  18. Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

    International Nuclear Information System (INIS)

    Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

    1987-01-01

    To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

  19. Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin.

    Science.gov (United States)

    Li, Sisi; Yen, Linda; Pastor, William A; Johnston, Jonathan B; Du, Jiamu; Shew, Colin J; Liu, Wanlu; Ho, Jamie; Stender, Bryan; Clark, Amander T; Burlingame, Alma L; Daxinger, Lucia; Patel, Dinshaw J; Jacobsen, Steven E

    2016-08-30

    Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.

  20. Isoform-Specific Na,K-ATPase Alterations Precede Disuse-Induced Atrophy of Rat Soleus Muscle

    Directory of Open Access Journals (Sweden)

    Violetta V. Kravtsova

    2015-01-01

    Full Text Available This study examines the isoform-specific effects of short-term hindlimb suspension (HS on the Na,K-ATPase in rat soleus muscle. Rats were exposed to 24–72 h of HS and we analyzed the consequences on soleus muscle mass and contractile parameters; excitability and the resting membrane potential (RMP of muscle fibers; the electrogenic activity, protein, and mRNA content of the α1 and α2 Na,K-ATPase; the functional activity and plasma membrane localization of the α2 Na,K-ATPase. Our results indicate that 24–72 h of HS specifically decreases the electrogenic activity of the Na,K-ATPase α2 isozyme and the RMP of soleus muscle fibers. This decrease occurs prior to muscle atrophy or any change in contractile parameters. The α2 mRNA and protein content increased after 24 h of HS and returned to initial levels at 72 h; however, even the increased content was not able to restore α2 enzyme activity in the disused soleus muscle. There was no change in the membrane localization of α2 Na,K-ATPase. The α1 Na,K-ATPase electrogenic activity, protein and mRNA content did not change. Our findings suggest that skeletal muscle use is absolutely required for α2 Na,K-ATPase transport activity and provide the first evidence that Na,K-ATPase alterations precede HS-induced muscle atrophy.

  1. Measurement of background translocation frequencies in individuals with clones

    Energy Technology Data Exchange (ETDEWEB)

    Wade, Marcelle J. [California State Univ. (CalState), Hayward, CA (United States)

    1996-08-01

    In the leukemia case the unseparated B and T lymphocytes had a high translocation frequency even after 0.0014, respectively. After purging all clones from the data, the translocation frequencies for Bio 8 and Bio 23 were 0.00750.0014 and 0.0073 metaphases were scored for chromosomal aberrations,, specifically reciprocal translocations, using fluorescence in situ hybridization (FISH). Metaphase spreads were used from two healthy, unexposed individuals (not exposed to radiation, chemotherapy or radiotherapy) and one early B- precursor acute lymphocytic leukemia (ALL) patient (metaphase spreads from both separated T lymphocytes and unseparated B and T lymphocytes were scored). All three individuals had an abnormally high translocation frequency. The high translocation frequencies resulted from clonal expansion of specific translocated chromosomes. I show in this thesis that by purging (discounting or removing) clones from the data of unexposed individuals, one can obtain true background translocation frequencies. In two cases, Bio 8 and Bio 23, the measured translocation frequency for chromosomes 1, 2 and 4 was 0.0124 purging all of the clones from the data. This high translocation frequency may be due to a low frequency of some clones and may not be recognized. The separated T lymphocytes had a higher translocation frequency than expected.

  2. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... resolution 3D structure the mechanism behind is only poorly understood. This thesis aimed at illuminating the autoinhibitory mechanism in plant and yeast PM H+-ATPases and below some of our main findings will be highlighted. The two terminal domains of the PM H+-ATPases have several amino acid residues...... that can be phosphorylated, and it has been demonstrated that these phosphorylation sites in both plant and yeast are highly involved in the regulation of terminal autoinhibition. In this study we used a phylogenetic analysis to investigate the evolutionary development of these phosphorylation sites...

  3. Enhanced diffusion due to electrons, protons and quenching

    International Nuclear Information System (INIS)

    Schuele, W.

    1987-01-01

    Results of investigations of radiation enhanced diffusion in copper -30% zinc alloys using 17.65 MeV protons are reported and compared with results obtained for 2 MeV electrons. The activation energy of diffusion decreases considerably from 0.35 eV to 0.26 eV for displacement rates increasing from 3x10 -12 dpa.s -1 to 1.2x10 -8 dpa.s -1 , i.e. the migration activation energy of interstitials decreases for this dpa.s -1 range from 0.70 eV to 0.52 eV. Results of electron irradiations obtained for 0.050 and 0.10 mm thick specimens are compared. It is found that the diffusion rates increase considerably in the presence of dislocations and that the diffusion rates decrease for very low electron fluxes and high irradiation temperatures in the 0.050 mm thick specimens in comparison to the rates obtained in 0.10 mm thick specimens. A value of 0.95 eV was determined for the activation energy of the ordering rate after quenching from 250 0 C in water. This was attributed to the migration activation energy of vacancies

  4. Characteristics of a (Na+K+)-ATPase inhibitor in extracts of tea

    International Nuclear Information System (INIS)

    Sagnella, G.A.; MacGregor, G.A.

    1984-01-01

    Extracts of tea were examined for inhibitors of the sodium-potassium pump by investigating the effect of the extracts on 1) isolated preparations of (Na + -K + )-ATPase from hog brain and human blood cells; 2) the displacement of radioactive ouabain from its specific receptor on red blood cells, and 3) the uptake of radioactive rubidium in intact red blood cells. It has been found that extracts of tea were potent inhibitors of the purified hog brain (Na + -K + )-ATPase. However, the inhibition was not specific for the (Na + -K + )-ATPase and the extract of tea did not displace 3 H-ouabain in a specific ouabain-receptor assay. Additionally, the tea extracts displayed only a small inhibitory effect on the uptake of 86 Rb in intact red blood cells. These observations suggest that the material is not like digitalis and that, unlike cardiac glycosides, it may inhibit the activity of the (Na + -K + )-ATPase by interacting with the enzyme at intracellular sites

  5. Translocations affecting human immunoglobulin heavy chain locus

    Directory of Open Access Journals (Sweden)

    Sklyar I. V.

    2014-03-01

    Full Text Available Translocations involving human immunoglobulin heavy chain (IGH locus are implicated in different leukaemias and lymphomas, including multiple myeloma, mantle cell lymphoma, Burkitt’s lymphoma and diffuse large B cell lymphoma. We have analysed published data and identified eleven breakpoint cluster regions (bcr related to these cancers within the IgH locus. These ~1 kbp bcrs are specific for one or several types of blood cancer. Our findings could help devise PCR-based assays to detect cancer-related translocations, to identify the mechanisms of translocations and to help in the research of potential translocation partners of the immunoglobulin locus at different stages of B-cell differentiation.

  6. β‑catenin nuclear translocation induced by HIF‑1α overexpression leads to the radioresistance of prostate cancer.

    Science.gov (United States)

    Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

    2018-04-12

    Hypoxia-inducible factor‑1α (HIF‑1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF‑1α remain unclear. β‑catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF‑1α and β‑catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4‑2B, were grouped as follows: Negative control (no treatment), HIF‑1α overexpression group (transfected with HIF‑1α overexpression plasmid) and β‑catenin silenced group (transfected with HIF‑1α plasmids and β‑catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4‑2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4‑2B cells, transfection with HIF‑1α overexpression plasmid led to an enhanced β‑catenin nuclear translocation, while β‑catenin silencing inhibited β‑catenin nuclear translocation. The enhanced β‑catenin nuclear translocation induced by HIF‑1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non‑homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF‑1α overexpression enhanced β‑catenin nuclear translocation, which led to the activation of the β‑catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF‑1α overexpression promotes the radioresistance of PCa cells.

  7. The ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering

    Science.gov (United States)

    Çamdere, Gamze; Guacci, Vincent; Stricklin, Jeremiah; Koshland, Douglas

    2015-01-01

    Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin’s DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition. DOI: http://dx.doi.org/10.7554/eLife.11315.001 PMID:26583750

  8. Vacuolar-type H+-ATPase and Na+, K+-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

    DEFF Research Database (Denmark)

    Seidelin, Michel; Brauner, Colin J; Jensen, Frank Bo

    2001-01-01

    Changes in branchial vacuolar-type H+-ATPase B-subunit mRNA and Na+, K+-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days...... Na+, K+-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H+-ATPase abundance as a compensatory...

  9. Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase.

    Science.gov (United States)

    Madan, Namrata; Xu, Yunhui; Duan, Qiming; Banerjee, Moumita; Larre, Isabel; Pierre, Sandrine V; Xie, Zijian

    2017-03-01

    The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na + affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na + was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1. Copyright © 2017 the American Physiological Society.

  10. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    Science.gov (United States)

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  11. A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage.

    Science.gov (United States)

    Bohnert, K Adam; Kenyon, Cynthia

    2017-11-30

    Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage. How the germline avoids transmitting damage from one generation to the next remains a fundamental question in biology. Here we identify a lysosomal switch that enhances germline proteostasis before fertilization. We find that Caenorhabditis elegans oocytes whose maturation is arrested by the absence of sperm exhibit hallmarks of proteostasis collapse, including protein aggregation. Remarkably, sperm-secreted hormones re-establish oocyte proteostasis once fertilization becomes imminent. Key to this restoration is activation of the vacuolar H + -ATPase (V-ATPase), a proton pump that acidifies lysosomes. Sperm stimulate V-ATPase activity in oocytes by signalling the degradation of GLD-1, a translational repressor that blocks V-ATPase synthesis. Activated lysosomes, in turn, promote a metabolic shift that mobilizes protein aggregates for degradation, and reset proteostasis by enveloping and clearing the aggregates. Lysosome acidification also occurs during Xenopus oocyte maturation; thus, a lysosomal switch that enhances oocyte proteostasis in anticipation of fertilization may be conserved in other species.

  12. Translocation of cell-penetrating peptides into Candida fungal pathogens.

    Science.gov (United States)

    Gong, Zifan; Karlsson, Amy J

    2017-09-01

    Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF) 3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF) 3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens. © 2017 The Protein Society.

  13. Use of proton-enhanced, natural abundance /sup 13/C NMR to study the molecular dynamics of model and biological membranes

    Energy Technology Data Exchange (ETDEWEB)

    Cornell, B A [Commonwealth Scientific and Industrial Research Organization, North Ryde (Australia). Div. of Food Research; Keniry, M [Sydney Univ. (Australia). Dept. of Physical Chemistry; Hiller, R G [Macquarie Univ., North Ryde (Australia). School of Biological Sciences; Smith, R [La Trobe Univ., Bundoora (Australia). Dept. of Biochemistry

    1980-06-16

    Proton-enhanced NMR of the natural abundance /sup 13/C nuclei is used to study the lipid mobility in dispersions containing cholesterol, the polypeptide gramicidin A, and in membrane proparations derived from spinach chloroplasts and bovine brain myelin.

  14. Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats.

    Science.gov (United States)

    Belcastro, A N; Turcotte, R; Rossiter, M; Secord, D; Maybank, P E

    1984-01-01

    The activation characteristics of Mg-ATP and Ca2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min-1 with an 8% incline. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 microM Ca2+ at pH 7.0 and 30 degrees C. For control skeletal muscle, at 1 and 10 microM Ca2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP (P less than 0.05). For E animals the myofibril ATPase activities at 10 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ the activities at 3 and 5 mM Mg-ATP were greater for the E animals (P less than 0.05). Increasing KCl concentrations resulted in greater inhibition for E animals. With cardiac muscle, the myofibril ATPase activities at 1.0 microM free Ca2+ were lower for E at all Mg-ATP levels (P less than 0.05). In contrast, at 10 microM Ca2+, the E group exhibited an elevated myofibril ATPase activity. The results indicate that Mg-ATP and Ca2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise.

  15. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    Science.gov (United States)

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

  16. Label Free Chromosome Translocation Detection with Silicon nanowires

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Andersen, Karsten Brandt; Frøhling, Kasper Bayer

    HROMOSOME translocation, which is a rearrangement of arms between two chromosomes, is a major group of chromosome abnormalities leading to cancer. As a result, two derivative chromosomes with sequences coming from both chromosomes are formed. The current translocation detection method is a Fluore......HROMOSOME translocation, which is a rearrangement of arms between two chromosomes, is a major group of chromosome abnormalities leading to cancer. As a result, two derivative chromosomes with sequences coming from both chromosomes are formed. The current translocation detection method...

  17. Stochastic resonance during a polymer translocation process

    International Nuclear Information System (INIS)

    Mondal, Debasish; Muthukumar, M.

    2016-01-01

    We have studied the occurrence of stochastic resonance when a flexible polymer chain undergoes a single-file translocation through a nano-pore separating two spherical cavities, under a time-periodic external driving force. The translocation of the chain is controlled by a free energy barrier determined by chain length, pore length, pore-polymer interaction, and confinement inside the donor and receiver cavities. The external driving force is characterized by a frequency and amplitude. By combining the Fokker-Planck formalism for polymer translocation and a two-state model for stochastic resonance, we have derived analytical formulas for criteria for emergence of stochastic resonance during polymer translocation. We show that no stochastic resonance is possible if the free energy barrier for polymer translocation is purely entropic in nature. The polymer chain exhibits stochastic resonance only in the presence of an energy threshold in terms of polymer-pore interactions. Once stochastic resonance is feasible, the chain entropy controls the optimal synchronization conditions significantly.

  18. Final Report for DE-FG02-04ER15626: P-type ATPases in Plants – Role of Lipid Flippases in Membrane Biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Harper, Jeffrey F. [Univ. of Nevada, Reno, NV (United States)

    2015-02-24

    The long-range goal of the research is to understand the structure and biological functions of different P-type ATPases (ion pumps) in plant cells, and to use that knowledge to enhance the production of bioenergy from plants, or plant-research inspired technologies. Ptype ATPases include ion pumps that specifically transport H+, Ca2+, Zn2+, Cu2+, K+, or Na+, as well as at least one unusual subfamily that appears to function as lipid flippases, flipping specific lipids from one side of a membrane bilayer to the other. As a group, P-type ATPases are thought to consume more than 1/3 of the cellular ATP in typical eukaryotic cells. Recent research in the Harper lab focused on understanding the biochemical and biological functions of P-type ATPases that flip lipids. These flippases belong to the P4 subfamily of P-type ATPases. The activity of lipid flippases is thought to induce membrane curvature and/or create an asymmetry in which certain lipid head groups are preferential exposed to one surface or the other. In Arabidopsis thaliana there are 12 members of this family referred to as Aminophospholipid ATPase (ALA) 1 to ALA12. Using genetic knockouts, the Harper lab has established that this unusual subfamily of P-type ATPases are critical for plants to cope with even modest changes in temperature (e.g., down to 15°C, or up to 30°C). In addition, members of one subclade are critical for cell expansion, and loss of function mutants result in severe dwarfism. Other members of this same sub-clade are critical for pollen tube growth, and loss of function mutants are sterile under conditions of hot days and cold nights. While the cellular processes that depend on lipid flippases are still unclear, the genetic analysis of loss of function mutants clearly show they are of fundamental importance to plant growth and response to the environment.

  19. H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

    Directory of Open Access Journals (Sweden)

    Ulfah Lutfiah

    2015-11-01

    Full Text Available Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA. This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2, and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159, and drastically lowered from the third to seventh day (0.289; 0.108; 0.062. Specific activities of H,K-ATPase was slightly higher in the first day (0.765, then was lowered in the third (0.685 and fifth day (0.655, and was higher in the seventh day (0.884. The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343. Specific activities of total CA was low in the first day (0.083, and was higher from the third to seventh day (0.111; 0.136; 0.144.Conclusion: In hypoxia

  20. Effects of phenol on ATPase activities in crude gill homogenates of rainbow trout (Salmo gairdneri Richardson)

    Energy Technology Data Exchange (ETDEWEB)

    Poston, T.M.

    1979-01-01

    The ATPase specific activities from crude gill homogenates of rainbow trout were lower than those from microsomal preparations reported in the literature. Sodium pump activity (ouabain sensitive NaK-ATPase) was demonstrable at 37/sup 0/C. An ouabain insensitive NaK-ATPase was demonstrable at temperatures below 30/sup 0/C and may represent a Na-ATPase activity reported by others. Energy of activation at 25/sup 0/C for total NaK-ATPase ws 10,500 cal.mole/sup -1/. Mg-baseline activity had an energy of activation at 25/sup 0/C of 15,600 cal.mole/sup -1/. Mg-baseline activity was thermally labile at temperatures in excess of 30/sup 0/C. Concentrations of Mg/sup +2/ in excess of 5 mM appeared to inhibit total NaK-ATPase activity. At 37/sup 0/C, Na/sup +/ and K/sup +/ exerted little, if any, stimulatory effect on ATPase activities, in spite of the fact that 37/sup 0/C was the only temperature at which sodium pump activity was demonstrable. MS-222 failed to produce any discernible changes in any of the demonstrable ATPase activities in crude gill homogenates. Total NaK-ATPase activities were more sensitive than Mg-baseline activities to in vitro inhibition by phenol. Concentrations of phenol which produce 50% inhibition in total NaK-ATPase produced only 35% inhibition in Mg-baseline activity. The nature of in vitro inhibition was uncompetitive. Sodium pump activity was unaffected by phenol at concentrations as high as 25 mM. An effort was made to demonstrate an in vivo effects of phenol on rainbow trout gill ATPase activites. An infestation of a parasite (Gyrodactilus) on the experimental fish precludes any definative assessment of in vivo effects.

  1. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    2010-02-01

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  2. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    Science.gov (United States)

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

  3. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

    Science.gov (United States)

    Clausen, Michael V; Hilbers, Florian; Poulsen, Hanne

    2017-01-01

    The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  4. Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase.

    Science.gov (United States)

    Ahrens, M L

    1981-04-06

    In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.

  5. Conflict bear translocation: investigating population genetics and fate of bear translocation in Dachigam National Park, Jammu and Kashmir, India.

    Science.gov (United States)

    Mukesh; Sharma, Lalit Kumar; Charoo, Samina Amin; Sathyakumar, Sambandam

    2015-01-01

    The Asiatic black bear population in Dachigam landscape, Jammu and Kashmir is well recognized as one of the highest density bear populations in India. Increasing incidences of bear-human interactions and the resultant retaliatory killings by locals have become a serious threat to the survivorship of black bears in the Dachigam landscape. The Department of Wildlife Protection in Jammu and Kashmir has been translocating bears involved in conflicts, henceforth 'conflict bears' from different sites in Dachigam landscape to Dachigam National Park as a flagship activity to mitigate conflicts. We undertook this study to investigate the population genetics and the fate of bear translocation in Dachigam National Park. We identified 109 unique genotypes in an area of ca. 650 km2 and observed bear population under panmixia that showed sound genetic variability. Molecular tracking of translocated bears revealed that mostly bears (7 out of 11 bears) returned to their capture sites, possibly due to homing instincts or habituation to the high quality food available in agricultural croplands and orchards, while only four bears remained in Dachigam National Park after translocation. Results indicated that translocation success was most likely to be season dependent as bears translocated during spring and late autumn returned to their capture sites, perhaps due to the scarcity of food inside Dachigam National Park while bears translocated in summer remained in Dachigam National Park due to availability of surplus food resources. Thus, the current management practices of translocating conflict bears, without taking into account spatio-temporal variability of food resources in Dachigam landscape seemed to be ineffective in mitigating conflicts on a long-term basis. However, the study highlighted the importance of molecular tracking of bears to understand their movement patterns and socio-biology in tough terrains like Dachigam landscape.

  6. Conflict bear translocation: investigating population genetics and fate of bear translocation in Dachigam National Park, Jammu and Kashmir, India.

    Directory of Open Access Journals (Sweden)

    Mukesh

    Full Text Available The Asiatic black bear population in Dachigam landscape, Jammu and Kashmir is well recognized as one of the highest density bear populations in India. Increasing incidences of bear-human interactions and the resultant retaliatory killings by locals have become a serious threat to the survivorship of black bears in the Dachigam landscape. The Department of Wildlife Protection in Jammu and Kashmir has been translocating bears involved in conflicts, henceforth 'conflict bears' from different sites in Dachigam landscape to Dachigam National Park as a flagship activity to mitigate conflicts. We undertook this study to investigate the population genetics and the fate of bear translocation in Dachigam National Park. We identified 109 unique genotypes in an area of ca. 650 km2 and observed bear population under panmixia that showed sound genetic variability. Molecular tracking of translocated bears revealed that mostly bears (7 out of 11 bears returned to their capture sites, possibly due to homing instincts or habituation to the high quality food available in agricultural croplands and orchards, while only four bears remained in Dachigam National Park after translocation. Results indicated that translocation success was most likely to be season dependent as bears translocated during spring and late autumn returned to their capture sites, perhaps due to the scarcity of food inside Dachigam National Park while bears translocated in summer remained in Dachigam National Park due to availability of surplus food resources. Thus, the current management practices of translocating conflict bears, without taking into account spatio-temporal variability of food resources in Dachigam landscape seemed to be ineffective in mitigating conflicts on a long-term basis. However, the study highlighted the importance of molecular tracking of bears to understand their movement patterns and socio-biology in tough terrains like Dachigam landscape.

  7. Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo

    2010-08-01

    The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

  8. The effect of acetylcholine on 14C-assimilates translocation of Isatis tinctoria L

    International Nuclear Information System (INIS)

    Yang Chongjun; Tang Feiyu; Zhang Ping; Guo Yuhai

    2004-01-01

    The effects of acetylcholine on 14 C-assimilates translocation are studied with source-channel-sink of Isatis tinctoria L. The experiments show that 0.01 mmol/L treatments of acetylcholine on the phloem, can improve the output of 14 C-assimilates in leaves indicating that acetylcholine enhances the activity of phloem transport. (authors)

  9. Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket.

    NARCIS (Netherlands)

    Koenderink, J.B.; Geibel, S.; Grabsch, E.; Pont, J.J.H.H.M. de; Bamberg, E.; Friedrich, T.

    2003-01-01

    Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp,

  10. Changes in acetylcholinesterase, Na+,K+-ATPase, and Mg2+-ATPase activities in the frontal cortex and the hippocampus of hyper- and hypothyroid adult rats.

    Science.gov (United States)

    Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

    2007-08-01

    The thyroid hormones (THs) are crucial determinants of normal development and metabolism, especially in the central nervous system. The metabolic rate is known to increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-adenosinetriphosphatase (ATPase) in the frontal cortex and the hippocampus of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, and hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. A region-specific behavior was observed concerning the examined enzyme activities in hyper- and hypothyroidism. In hyperthyroidism, AChE activity was significantly increased only in the hippocampus (+22%), whereas Na+,K+-ATPase activity was significantly decreased in the hyperthyroid rat hippocampus (-47%) and remained unchanged in the frontal cortex. In hypothyroidism, AChE activity was significantly decreased in the frontal cortex (-23%) and increased in the hippocampus (+21%). Na+,K+-ATPase activity was significantly decreased in both the frontal cortex (-35%) and the hippocampus (-43%) of hypothyroid rats. Mg2+-ATPase remained unchanged in the regions of both hyper- and hypothyroid rat brains. Our data revealed that THs affect the examined adult rat brain parameters in a region- and state-specific way. The TH-reduced Na+,K+-ATPase activity may increase the synaptic acetylcholine release and, thus, modulate AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems in the examined brain regions.

  11. Radionuclide assay of membrane Na+, K+-ATPase activity of peserved red blood cells

    International Nuclear Information System (INIS)

    Trusov, V.V.; Zelenin, A.A.; Marizin, S.A.

    1986-01-01

    The radionuclide tests were used to investigate the influence of varying blood preservatives on erythrocylic membrane Na + , K + -ATPase activity in samples of whole blood and packed red blood cells from normal donors prepared by standard methods. The tests were performed before and after seven days of preservation under standard conditions. It was found that blood preservations lowered membrane Na + , K + -ATPase activity: its minimum reduction was recorded with citroglucopnosphate, while glugicir induced a significant drop in Na + , K + -ATPase activity of preserved red blood cells regardless of the type of the blood transfusion solution. The assay of membrane Na + , K + -ATPase activity of preserved red blood cells with the use of 86 Rb could be recommended as an evaluation test for preserved blood and its components

  12. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

  13. 11C-methionine translocation in barley

    International Nuclear Information System (INIS)

    Nakanishi, Hiromi; Bughio, Naimatullah; Shigeta Ishioka, Noriko

    2000-01-01

    11 C-methionine was supplied to barley plants through a single leaf or via the roots and real time 11 C movement was monitored using a PETIS (positron emitting tracer imaging system). In Fe-deficient plants, 11 C-methionine was translocated from the tip of the absorbing leaf to the discrimination center' at the basal part of the shoot and then retranslocated to all the chlorotic leaves, while a negligible amount was retranslocated to the roots. In Fe-sufficient plants, methionine was translocated from the absorbing leaf to the discrimination center and then only to the newest leaf on the main shoot. A negligible amount was also retranslocated to the roots. Although, in Fe-sufficient plants, methionine translocation was observed from absorbing roots to shoots, in Fe-deficient plants, only a little amount was translocated from roots to shoots. In conclusion, methionine from the upper portion of a plant is not used as a precursor of mugineic acid under Fe-deficiency conditions. (author)

  14. Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity

    Science.gov (United States)

    Mcdonald, K. S.; Fitts, R. H.

    1993-01-01

    The effect of hindlimb unweighting (HU) for 1 to 3 wks on the shortening velocity of a soleus fiber, its ATPase content, and the relative contents of the slow and fast myosin was investigated by measuring fiber force, V(0), ATPase activity, and myosin content in SDS protein profiles of a single rat soleus fiber suspended between a motor arm and a transducer. It was found that HU induces a progressive increase in fiber V(0) that is likely caused, at least in part, by an increase in the fiber's myofibrillar ATPase activity. The HU-induced increases in V(0) and ATPase were associated with the presence of a greater percentage of fast type IIa fibers. However, a large population of fibers after 1, 2, and 3 wks of HU showed increases in V(0) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers.

  15. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

    Directory of Open Access Journals (Sweden)

    Michael V. Clausen

    2017-06-01

    Full Text Available The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  16. Data on Na,K-ATPase in primary cultures of renal proximal tubule cells treated with catecholamines

    Directory of Open Access Journals (Sweden)

    Mary Taub

    2016-03-01

    Full Text Available This data article is concerned with chronic regulation of Na,K-ATPase by catecholamines. After a chronic treatment, inhibition of Na,K-ATPase activity was observed in cultures with dopamine, while a stimulation was observed in cultures treated with norepinephrine. Following a chronic incubation with guanabenz, an α adrenergic agonist, an increase in Na,K-ATPase α and β subunit mRNAs was observed. This data supports the research article entitled, “Renal proximal tubule Na, K-ATPase is controlled by CREB regulated transcriptional coactivators as well as salt inducible kinase 1” (Taub et al. 2015 [1]. Keywords: Catecholamines, Kidney, Proximal tubule, Na,K-ATPase, Chronic

  17. Nucleotide binding to Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  18. Increased leucocyte Na-K ATPase in obesity: reversal following weight loss

    International Nuclear Information System (INIS)

    Turaihi, K.; Baron, D.N.; Dandona, P.

    1987-01-01

    Ouabain-sensitive 86 Rb influx and [ 3 H] ouabain binding capacity were investigated in the leucocytes of 17 obese patients and 15 control subjects. Both were significantly increased in the obese when compared with controls. Following dietary restriction and a 4% to 5% weight reduction in the obese over 2 weeks, [ 3 H] ouabain binding and ouabain-sensitive 86 Rb influx (a model for K+ influx) decreased to levels similar to those in controls. This shows that the number of Na-K ATPase sites on leucocyte membranes of the obese are significantly increased and that this is associated with accelerated 86 Rb transport. Since both of these indices decreased following 4% to 5% reduction in body weight while the patients were still obese, increased Na-K ATPase is neither a marker of nor cardinal to the pathogenesis of obesity. We conclude that (1) increase in Na-K ATPase units and 86 Rb influx are not characteristic of obesity itself and (2) dietary restriction over the short-term with limited weight reduction restores Na-K ATPase units and 86 Rb influx to normal

  19. Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain.

    Science.gov (United States)

    Miles, Andrew J; Fedosova, Natalya U; Hoffmann, Søren V; Wallace, B A; Esmann, Mikael

    2013-05-31

    Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography. Copyright © 2013 The Author. Published by Elsevier Inc. All rights reserved.

  20. (+-Rutamarin as a dual inducer of both GLUT4 translocation and expression efficiently ameliorates glucose homeostasis in insulin-resistant mice.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    Full Text Available Glucose transporter 4 (GLUT4 is a principal glucose transporter in response to insulin, and impaired translocation or decreased expression of GLUT4 is believed to be one of the major pathological features of type 2 diabetes mellitus (T2DM. Therefore, induction of GLUT4 translocation or/and expression is a promising strategy for anti-T2DM drug discovery. Here we report that the natural product (+-Rutamarin (Rut functions as an efficient dual inducer on both insulin-induced GLUT4 translocation and expression. Rut-treated 3T3-L1 adipocytes exhibit efficiently enhanced insulin-induced glucose uptake, while diet-induced obese (DIO mice based assays further confirm the Rut-induced improvement of glucose homeostasis and insulin sensitivity in vivo. Subsequent investigation of Rut acting targets indicates that as a specific protein tyrosine phosphatase 1B (PTP1B inhibitor Rut induces basal GLUT4 translocation to some extent and largely enhances insulin-induced GLUT4 translocation through PI3 kinase-AKT/PKB pathway, while as an agonist of retinoid X receptor α (RXRα, Rut potently increases GLUT4 expression. Furthermore, by using molecular modeling and crystallographic approaches, the possible binding modes of Rut to these two targets have been also determined at atomic levels. All our results have thus highlighted the potential of Rut as both a valuable lead compound for anti-T2DM drug discovery and a promising chemical probe for GLUT4 associated pathways exploration.