Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing.

Science.gov (United States)

Snoberger, Aaron; Brettrager, Evan J; Smith, David M

2018-06-18

Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN's symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases' coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils regulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity.

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.

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.

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.

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.

Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase

Directory of Open Access Journals (Sweden)

Forest H. Andrews

2016-09-01

Full Text Available MORC3 is linked to inflammatory myopathies and cancer; however, the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA, but it is negatively regulated by the CW domain, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing MORC3 functions and suggest that its modulation may contribute to human disease.

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.

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

Science.gov (United States)

Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

2017-03-17

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

Science.gov (United States)

Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

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...

The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

Science.gov (United States)

Chen, Kelan; Dobson, Renwick C J; Lucet, Isabelle S; Young, Samuel N; Pearce, F Grant; Blewitt, Marnie E; Murphy, James M

2016-06-15

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Extracellular Na+ levels regulate formation and activity of the NaX/alpha1-Na+/K+-ATPase complex in neuronal cells.

Directory of Open Access Journals (Sweden)

Emmanuelle eBerret

2014-12-01

Full Text Available MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na+]out. The mechanism underlying Na+-sensing involves Na+-flow through the NaX channel, directly regulated by the Na+/K+-ATPase α1-isoform which controls Na+-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na+]in. Here we aim to determine whether environmental changes in Na+ could actively modulate the NaX/Na+/K+-ATPase complex activity.We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na+] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na+] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na+/K+-ATPase remained unaltered. This unbalance between NaX and Na+/K+-ATPase pump proportion would induce a bigger portion of Na+/K+-ATPase-control-free NaX channel. Thus we suggest that hypernatremic environment increases NaX/Na+/K+-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.

Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

DEFF Research Database (Denmark)

Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

2005-01-01

In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

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

Science.gov (United States)

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.

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

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

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.

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.

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...

A marked animal-vegetal polarity in the localization of Na(+),K(+) -ATPase activity and its down-regulation following progesterone-induced maturation.

Science.gov (United States)

Mohanty, Basant Kumar; Gupta, Brij L

2012-02-01

The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. In this study, we show the expression and distribution pattern of Na(+),K(+) -ATPase in stage-VI oocytes, and its changes following progesterone-induced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [(3) H]-ouabain autoradiography, and immunofluorescence cytochemistry at light microscopic level, we find that Na(+),K(+) -ATPase activity is mainly confined to the animal hemisphere. Electron microscopy histochemical results also suggest that polarized distribution of Na(+),K(+) -ATPase activity persists following progesterone-induced maturation, and it becomes gradually more polarized towards the animal pole. The time course following progesterone-induced maturation suggests that there is an initial up-regulation and then gradual down-regulation of Na(+),K(+) -ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na(+),K(+) -ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte. This study provides the first direct evidence for a marked asymmetric localization of Na(+),K(+) -ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na(+),K(+) -ATPase activity in stage-VI oocytes, and their down-regulation following progesterone-induced maturation, is likely to have a role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD. Copyright © 2011 Wiley Periodicals, Inc.

Lens ion transport: from basic concepts to regulation of Na,K-ATPase activity

Science.gov (United States)

Delamere, Nicholas A.; Tamiya, Shigeo

2009-01-01

In the late 1960s, studies by George Duncan explained many of the basic principles that underlie lens ion homeostasis. The experiments pointed to a permeability barrier close to the surface of the lens and illustrated the requirement for continuous Na,K-ATPase-mediated active sodium extrusion. Without active sodium extrusion, lens sodium and calcium content increases resulting in lens swelling and deterioration of transparency. Later, Duncan's laboratory discovered functional muscarinic and purinergic receptors at the surface of the lens. Recent studies using intact lens suggest purinergic receptors might be involved in short-term regulation of Na,K-ATPase in the epithelium. Purinergic receptor agonists ATP and UTP selectively activate certain Src family tyrosine kinases and stimulate Na,K-ATPase activity. This might represent part of a control mechanism capable of adjusting, perhaps fine tuning, lens ion transport machinery. PMID:18614168

Role of protein kinase C in regulation of Na+- and K +-dependent ATPase activity and pump function in corneal endothelial cells.

Science.gov (United States)

Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Nishida, Teruo

2009-05-01

Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, 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 a 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. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.

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...

RIN4 functions with plasma membrane H+-ATPases to regulate stomatal apertures during pathogen attack

DEFF Research Database (Denmark)

Liu, Jun; Elmore, James M.; Fuglsang, Anja Thoe

2009-01-01

Abstract Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4- mediated immune signal transduction, we...... purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H+-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines...... exhibit differential PM H+-ATPase activity. PM H+-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H+-ATPase activity and, importantly, its...

Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1) ATP complex

International Nuclear Information System (INIS)

Greene, L.E.; Williams, D.L. Jr.; Eisenberg, E.

1987-01-01

In the authors' model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1 ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca 2+ , S-1 ATP should not turn on the regulated actin filament. In the present study, they tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM S-1) so that functionally it acts like S-1 ATP, although it does not hydrolyze ATP. [ 14 C]pPDM and [ 32 P]ATP were used as tracers. They found that, in the absence of Ca 2+ , neither S-1 ATP nor pPDM S-1 ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto S-1). In contrast, in the presence of Ca 2+ , pPDM S-1 ATP binding almost completely turns on the regulated acto S-1 ATPase activity. These results can be explained by their original cooperativity model, with pPDM S-1 ATP binding only ≅ 2 fold more strongly to the turned-on form that to the turned-off form of regulated actin. However, the results are not consistent with our alternative model, which predicts that if pPDM S-1 ATP binds to actin in the absence of Ca 2+ but does not turn on the ATPase activity, then it should also turn on the ATPase activity in the presence of Ca 2+

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

Directory of Open Access Journals (Sweden)

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.

Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase

DEFF Research Database (Denmark)

Jorgensen, P.L.

2001-01-01

genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular...... mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites....

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.

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

Science.gov (United States)

Alves, Daiane S; Farr, Glen A; Seo-Mayer, Patricia; Caplan, Michael J

2010-12-01

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

Na+ pump in renal tubular cells is regulated by endogenous Na+-K+-ATPase inhibitor from hypothalamus

International Nuclear Information System (INIS)

Cantiello, H.F.; Chen, E.; Ray, S.; Haupert, G.T. Jr.

1988-01-01

Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na + -K + -ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK 1 ) were studied using 86 Rb + uptake and [ 3 H]ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na + -K + -ATPase in LLC-PK 1 cells. In contrast to membrane studies, no prolonged incubation with LLC-PK 1 was needed to observe inhibition of Na + -K + -ATPase. HF caused a 33% inhibition of ouabain-sensitive 86 Rb + influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK 1 86 Rb + uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na + -K + -ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes

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....

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

Science.gov (United States)

Case, Heather B; Dickenson, Nicholas E

2018-04-17

Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. In vitro and in vivo characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in Shigella T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how Shigella controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein.

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.

Na,K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase dependent connexin43 phosphorylation

DEFF Research Database (Denmark)

Hangaard, Lise; Bouzinova, Elena; Stæhr, Christian Albeck

2017-01-01

Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We...... coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall...

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

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

Science.gov (United States)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4

OpenAIRE

Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

2012-01-01

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which prov...

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.

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

Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4.

Science.gov (United States)

Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

2012-07-30

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase.

Science.gov (United States)

Namkoong, Sim; Lee, Kang Il; Lee, Jin I; Park, Rackhyun; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

2015-01-01

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

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.

Thyroid hormone coordinately regulates Na sup + -K sup + -ATPase. alpha. - and. beta. -subunit mRNA levels in kidney

Energy Technology Data Exchange (ETDEWEB)

McDonough, A.A.; Brown, T.A.; Horowitz, B.; Chiu, R.; Schlotterbeck, J.; Bowen, J.; Schmitt, C.A. (Univ. of Southern California School of Medicine, Los Angeles (USA))

1988-02-01

Synthesis of the sodium pump, Na{sup +}-K{sup +}-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T{sub 3}) regulates the concentration of the mRNAs coding for the two enzyme subunits, {alpha} and {beta}, and the time course of the response. A single dose of T{sub 3} was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T{sub 3}-injected animals, as well as hypothyroid and euthyroid rats, {alpha}- and {beta}-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; {alpha}-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na{sup +}-K{sup +}-ATPase activity was measured enzymatically. {alpha}- and {beta}-mRNAs increased coordinately to 1.6-fold over hypothyroid levels by 12 h after T{sub 3}. The authors conclude that T{sub 3} regulates Na{sup +}-K{sup +}-ATPase synthesis and activity by coordinately increasing the mRNAs of both the {alpha}- and {beta}-subunits of the enzyme.

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.

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

Science.gov (United States)

Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

1999-07-01

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

NARCIS (Netherlands)

L.J. Blok (Leen); G.T.G. Chang; M. Steenbeek-Slotboom (M.); W.M. van Weerden (Wytske); H.G. Swarts; J.J.H.H.M. de Pont (J. J H H M); G.J. van Steenbrugge (Gert Jan); A.O. Brinkmann (Albert)

1999-01-01

textabstractThe β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of

Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+, K+-ATPase activity

International Nuclear Information System (INIS)

Angel, I.; Hauger, R.L.; Luu, M.D.; Giblin, B.; Skolnick, P.; Paul, S.M.

1985-01-01

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 0 C) resulted in a time-dependent decrease in specific (+)-[ 3 H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[ 3 H]amphetamine binding, whereas incubations with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[ 3 H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[ 3 H]amphetamine binding, suggesting the involvement of Na + , K + -ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na + ,K + -ATPase activity and the number of specific high-affinity binding sites for [ 3 H]ouabain, and a good correlation was observed between the glucose-stimulated increase in (+)-[ 3 H]amphetamine and [ 3 H]ouabain binding. These data suggest that the (+)-[ 3 H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na + ,K + -ATPase activity, and the latter may be involved in the glucostatic regulation of appetite

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1

NARCIS (Netherlands)

Prattes, M.; Loibl, M.; Zisser, G.; Luschnig, D.; Kappel, L.; Rossler, I.; Grassegger, M.; Hromic, A.; Krieger, E.; Gruber, K.; Pertschy, B.; Bergler, H.

2017-01-01

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP

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.

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.

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.

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

Science.gov (United States)

Alves, Daiane S.; Thulin, Gunilla; Loffing, Johannes; Kashgarian, Michael

2015-01-01

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na+,K+-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na+,K+-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na+,K+-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na+,K+-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion–induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na+,K+-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na+,K+-ATPase to the energy state of renal epithelial cells. PMID:25788531

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia.

Science.gov (United States)

Alves, Daiane S; Thulin, Gunilla; Loffing, Johannes; Kashgarian, Michael; Caplan, Michael J

2015-11-01

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na(+),K(+)-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na(+),K(+)-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na(+),K(+)-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na(+),K(+)-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion-induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na(+),K(+)-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na(+),K(+)-ATPase to the energy state of renal epithelial cells. Copyright © 2015 by the American Society of Nephrology.

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.

Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+ -ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

DEFF Research Database (Denmark)

Madsen, Steffen; Jensen, Lars Nørholm; Tipsmark, Christian Kølbaek

2007-01-01

-regulated kinase (ERK) 1/2 was stimulated by EGF but not affected by IGF-I. This study is the first to report a branchial EGF response and to demonstrate a functional ERK 1/2 pathway in the teleost gill. In conclusion, CFTR and Na(+),K(+) -ATPase are differentially regulated by salinity and hormones in gills...

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.

Na sup + pump in renal tubular cells is regulated by endogenous Na sup + -K sup + -ATPase inhibitor from hypothalamus

Energy Technology Data Exchange (ETDEWEB)

Cantiello, H.F.; Chen, E.; Ray, S.; Haupert, G.T. Jr. (Harvard medical School, Boston, MA (USA))

1988-10-01

Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na{sup +}-K{sup +}-ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK{sup 1}) were studied using {sup 86}Rb{sup +} uptake and ({sup 3}H)ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na{sup +}-K{sup +}-ATPase in LLC-PK{sub 1} cells. In contrast to membrane studies, no prolonged incubation with LLC-PK{sub 1} was needed to observe inhibition of Na{sup +}-K{sup +}-ATPase. HF caused a 33% inhibition of ouabain-sensitive {sup 86}Rb{sup +} influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK{sub 1} {sup 86}Rb{sup +} uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na{sup +}-K{sup +}-ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes.

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/

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/.

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....

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...

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).

Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms.

Science.gov (United States)

Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

2017-11-01

Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation. © 2017 Wiley Periodicals, Inc.

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.

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...

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

Novel Role for Na,K-ATPase in Phosphatidylinositol 3-Kinase Signaling and Suppression of Cell Motility

OpenAIRE

Barwe, Sonali P.; Anilkumar, Gopalakrishnapillai; Moon, Sun Y.; Zheng, Yi; Whitelegge, Julian P.; Rajasekaran, Sigrid A.; Rajasekaran, Ayyappan K.

2005-01-01

The Na,K-ATPase, consisting of α- and β-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase β1-subunit (Na,K-β) in highly motile Moloney sarcoma virus-transforme...

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

C. pneumoniae CdsL regulates CdsN ATPase activity, and disruption with a peptide mimetic prevents bacterial invasion

Directory of Open Access Journals (Sweden)

Chris Blair Stone

2011-02-01

Full Text Available Chlamydiae are obligate intracellular pathogens that likely require type III secretion (T3S to invade cells and replicate intracellulary within a cytoplasmic vacuole called an inclusion body. C. pneumoniae possess a YscL ortholog, CdsL, that has been shown to interact with the T3S ATPase (CdsN. In this report we demonstrate that CdsL down-regulates CdsN enzymatic activity in a dose-dependent manner. Using PepScan epitope mapping we identified two separate binding domains to which CdsL binds viz. CdsN 221-229 and CdsN265-270. We confirmed the binding domains using a pull-down assay and showed that GST-CdsN221-270, which encompasses these peptides, co-purified with His-CdsL. Next, we used orthology modeling based on the crystal structure of a T3S ATPase ortholog from E. coli, EscN, to map the binding domains on the predicted three dimensional structure of CdsN. The CdsL binding domains mapped to the catalytic domain of the ATPase, one in the central channel of the ATPase hexamer and one on the outer face. Since peptide mimetics have been used to disrupt essential protein interactions of the chlamydial T3S system and inhibit T3S-mediated invasion of HeLa cells, we hypothesized that if CdsL – CdsN binding is essential for regulating T3S then a CdsN peptide mimetic could be used to potentially block T3S and Chlamydial invasion. Treatment of EBs with a CdsN peptide mimetic inhibited C. pneumoniae invasion into HeLa cells in a dose-dependent fashion. This report represents the first use of Pepscan technology to identify binding domains for specific T3S proteins viz. CdsL on the ATPase, CdsN, and demonstrates that peptide mimetics can be used as anti-virulence factors to block bacterial invasion.

Neuromuscular regulation in zebrafish by a large AAA+ ATPase/ubiquitin ligase, mysterin/RNF213

Science.gov (United States)

Kotani, Yuri; Morito, Daisuke; Yamazaki, Satoru; Ogino, Kazutoyo; Kawakami, Koichi; Takashima, Seiji; Hirata, Hiromi; Nagata, Kazuhiro

2015-01-01

Mysterin (also known as RNF213) is a huge intracellular protein with two AAA+ ATPase modules and a RING finger ubiquitin ligase domain. Mysterin was originally isolated as a significant risk factor for the cryptogenic cerebrovascular disorder moyamoya disease, and was found to be involved in physiological angiogenesis in zebrafish. However, the function and the physiological significance of mysterin in other than blood vessels remain largely unknown, although mysterin is ubiquitously expressed in animal tissues. In this study, we performed antisense-mediated suppression of a mysterin orthologue in zebrafish larvae and revealed that mysterin-deficient larvae showed significant reduction in fast myofibrils and immature projection of primary motoneurons, leading to severe motor deficits. Fast muscle-specific restoration of mysterin expression cancelled these phenotypes, and interestingly both AAA+ ATPase and ubiquitin ligase activities of mysterin were indispensable for proper fast muscle formation, demonstrating an essential role of mysterin and its enzymatic activities in the neuromuscular regulation in zebrafish. PMID:26530008

Regulation of Na(+)/K(+)-ATPase by nuclear respiratory factor 1: implication in the tight coupling of neuronal activity, energy generation, and energy consumption.

Science.gov (United States)

Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

2012-11-23

NRF-1 regulates mediators of neuronal activity and energy generation. NRF-1 transcriptionally regulates Na(+)/K(+)-ATPase subunits α1 and β1. NRF-1 functionally regulates mediators of energy consumption in neurons. NRF-1 mediates the tight coupling of neuronal activity, energy generation, and energy consumption at the molecular level. Energy generation and energy consumption are tightly coupled to neuronal activity at the cellular level. Na(+)/K(+)-ATPase, a major energy-consuming enzyme, is well expressed in neurons rich in cytochrome c oxidase, an important enzyme of the energy-generating machinery, and glutamatergic receptors that are mediators of neuronal activity. The present study sought to test our hypothesis that the coupling extends to the molecular level, whereby Na(+)/K(+)-ATPase subunits are regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), found recently by our laboratory to regulate all cytochrome c oxidase subunit genes and some NMDA and AMPA receptor subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutational analysis, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Atp1a1 and Atp1b1 genes but not of the Atp1a3 gene in neurons. The transcripts of Atp1a1 and Atp1b1 subunit genes were up-regulated by KCl and down-regulated by tetrodotoxin. Atp1b1 is positively regulated by NRF-1, and silencing of NRF-1 with small interference RNA blocked the up-regulation of Atp1b1 induced by KCl, whereas overexpression of NRF-1 rescued these transcripts from being suppressed by tetrodotoxin. On the other hand, Atp1a1 is negatively regulated by NRF-1. The binding sites of NRF-1 on Atp1a1 and Atp1b1 are conserved among mice, rats, and humans. Thus, NRF-1 regulates key Na(+)/K(+)-ATPase subunits and plays an important role in mediating the tight coupling between

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.

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.

Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Directory of Open Access Journals (Sweden)

Claudia Colina

2010-11-01

Full Text Available Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+/K(+ pumps in order to maintain ion homeostasis. In this study we show that Na(+/K(+ pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+/K(+ ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+ release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

Role of the plasma membrane H(+)-ATPase in the regulation of organic acid exudation under aluminum toxicity and phosphorus deficiency.

Science.gov (United States)

Yu, Wenqian; Kan, Qi; Zhang, Jiarong; Zeng, Bingjie; Chen, Qi

2016-01-01

Aluminum (Al) toxicity and phosphorus (P) deficiency are 2 major limiting factors for plant growth and crop production in acidic soils. Organic acids exuded from roots have been generally regarded as a major resistance mechanism to Al toxicity and P deficiency. The exudation of organic acids is mediated by membrane-localized OA transporters, such as ALMT (Al-activated malate transporter) and MATE (multidrug and toxic compound extrusion). Beside on up-regulation expression of organic acids transporter gene, transcriptional, translational and post-translational regulation of the plasma membrane H(+)-ATPase are also involved in organic acid release process under Al toxicity and P deficiency. This mini-review summarizes the current knowledge about this field of study on the role of the plasma membrane H(+)-ATPase in organic acid exudation under Al toxicity and P deficiency conditions.

Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.

Science.gov (United States)

Abu-Remaileh, Monther; Wyant, Gregory A; Kim, Choah; Laqtom, Nouf N; Abbasi, Maria; Chan, Sze Ham; Freinkman, Elizaveta; Sabatini, David M

2017-11-10

The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H + -adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

Science.gov (United States)

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

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.

Role of the plasma membrane H+-ATPase in the regulation of organic acid exudation under aluminum toxicity and phosphorus deficiency

Science.gov (United States)

Yu, Wenqian; Kan, Qi; Zhang, Jiarong; Zeng, Bingjie; Chen, Qi

2016-01-01

Aluminum (Al) toxicity and phosphorus (P) deficiency are 2 major limiting factors for plant growth and crop production in acidic soils. Organic acids exuded from roots have been generally regarded as a major resistance mechanism to Al toxicity and P deficiency. The exudation of organic acids is mediated by membrane-localized OA transporters, such as ALMT (Al-activated malate transporter) and MATE (multidrug and toxic compound extrusion). Beside on up-regulation expression of organic acids transporter gene, transcriptional, translational and post-translational regulation of the plasma membrane H+-ATPase are also involved in organic acid release process under Al toxicity and P deficiency. This mini-review summarizes the current knowledge about this field of study on the role of the plasma membrane H+-ATPase in organic acid exudation under Al toxicity and P deficiency conditions. PMID:26713714

Na/K-ATPase Signaling and Salt Sensitivity: The Role of Oxidative Stress

Directory of Open Access Journals (Sweden)

Jiang Liu

2017-03-01

Full Text Available Other than genetic regulation of salt sensitivity of blood pressure, many factors have been shown to regulate renal sodium handling which contributes to long-term blood pressure regulation and have been extensively reviewed. Here we present our progress on the Na/K-ATPase signaling mediated sodium reabsorption in renal proximal tubules, from cardiotonic steroids-mediated to reactive oxygen species (ROS-mediated Na/K-ATPase signaling that contributes to experimental salt sensitivity.

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

Excessive Extracellular ATP Desensitizes P2Y2 and P2X4 ATP Receptors Provoking Surfactant Impairment Ending in Ventilation-Induced Lung Injury

Directory of Open Access Journals (Sweden)

Djo Hasan

2018-04-01

Full Text Available Stretching the alveolar epithelial type I (AT I cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP (purinergic signaling. Extracellular ATP is cleared by extracellular ATPases, maintaining its homeostasis and enabling the lung to adapt the exocytosis of surfactant to the demand. Vigorous deformation of the AT I cells by high mechanical power ventilation causes a massive release of extracellular ATP beyond the clearance capacity of the extracellular ATPases. When extracellular ATP reaches levels >100 μM, the ATP receptors of the AT II cells become desensitized and surfactant impairment is initiated. The resulting alteration in viscoelastic properties and in alveolar opening and collapse time-constants leads to alveolar collapse and the redistribution of inspired air from the alveoli to the alveolar ducts, which become pathologically dilated. The collapsed alveoli connected to these dilated alveolar ducts are subject to a massive strain, exacerbating the ATP release. After reaching concentrations >300 μM extracellular ATP acts as a danger-associated molecular pattern, causing capillary leakage, alveolar space edema, and further deactivation of surfactant by serum proteins. Decreasing the tidal volume to 6 mL/kg or less at this stage cannot prevent further lung injury.

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

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....

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.

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

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

V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus.

Directory of Open Access Journals (Sweden)

Peng Lü

Full Text Available Silkworms are usually susceptible to the infection of Bombyx mori (B. mori nucleopolyhedrovirus (BmNPV, which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells of BmNPV-resistant strains (NB and BC8, the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi, the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.

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

Regulation of potassium dependent ATPase (kdp) operon of Deinococcus radiodurans.

Science.gov (United States)

Dani, Pratiksha; Ujaoney, Aman Kumar; Apte, Shree Kumar; Basu, Bhakti

2017-01-01

The genome of D. radiodurans harbors genes for structural and regulatory proteins of Kdp ATPase, in an operon pattern, on Mega plasmid 1. Organization of its two-component regulatory genes is unique. Here we demonstrate that both, the structural as well as regulatory components of the kdp operon of D. radiodurans are expressed quickly as the cells experience potassium limitation but are not expressed upon increase in osmolarity. The cognate DNA binding response regulator (RR) effects the expression of kdp operon during potassium deficiency through specific interaction with the kdp promoter. Deletion of the gene encoding RR protein renders the mutant D. radiodurans (ΔRR) unable to express kdp operon under potassium limitation. The ΔRR D. radiodurans displays no growth defect when grown on rich media or when exposed to oxidative or heat stress but shows reduced growth following gamma irradiation. The study elucidates the functional and regulatory aspects of the novel kdp operon of this extremophile, for the first time.

Insulin regulation of (Na+, K+)-ATPase

International Nuclear Information System (INIS)

Lytton, J.

1985-01-01

This thesis describes an investigation into the mechanism of insulin stimulation of (Na + ,K + )=ATPase in rat adipocytes. Two molecular forms of the catalytic subunit of the enzyme were identified and denoted α and α(+), due to their similarity to those isozymes previously described from rat brain. Insulin specifically stimulated the α(+) form of the enzyme. The two forms of the enzyme had quite different affinities for intracellular sodium ion; insulin affected only the lower affinity of α(+), shifting it toward a higher value. However, the sodium affinity of (Na + ,K + )-ATPase activity in isolated membranes was equally high for both forms of the enzyme. This suggests that the difference in sodium affinity between the two forms observed in the cell is not inherent within the structure of the sodium pump, but must depend upon a selective interaction with another molecule which has been lost upon membrane isolation. Immunoprecipitation of both the catalytic subunits either from extracts of whole cells which had been labelled with [ 32 P] orthophosphate, or from membranes which had been labelled with γ-[ 32 P]ATP demonstrated that less than 1 in 100 molecules had a covalently bound phosphate insulin had no influence on this value. The amino terminal sequences of the first 4 amino acids of the catalytic subunits of both α (isolated from rat kidney) and α(+) (from rat brainstem axolemma) were determined. The result shows two highly homologous but clearly different molecules. It can thus be concluded that the insulin sensitive version of the enzyme is not derived from the common α form by a post-translational modification

Excessive extracellular ATP desensitizes P2Y2 and P2X4 ATP receptors provoking surfactant impairment ending in ventilation-induced lung injury

NARCIS (Netherlands)

D. Hasan (Djo); Satalin, J. (Joshua); van der Zee, P. (Philip); Kollisch-Singule, M. (Michaela); P. Blankman (Paul); Shono, A. (Atsuko); P. Somhorst (Peter); C.A. den Uil (Corstiaan); H.J. Meeder (Han); Kotani, T. (Toru); G.F. Nieman (Gary F.)

2018-01-01

textabstractStretching the alveolar epithelial type I (AT I) cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP) (purinergic signaling). Extracellular ATP is cleared by extracellular ATPases,

Salt-induced Na+/K+-ATPase-α/β expression involves soluble adenylyl cyclase in endothelial cells.

Science.gov (United States)

Mewes, Mirja; Nedele, Johanna; Schelleckes, Katrin; Bondareva, Olga; Lenders, Malte; Kusche-Vihrog, Kristina; Schnittler, Hans-Joachim; Brand, Stefan-Martin; Schmitz, Boris; Brand, Eva

2017-10-01

High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca 2+ - and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na + /K + -ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na + )-induced Na + /K + -ATPase-α and Na + /K + -ATPase-β protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na + /K + -ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na + /K + -ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na + /K + -ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na + /K + -ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na + /K + -ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

The role of Na,K-ATPase/Src-kinase signaling pathway in the vascular wall contaction

DEFF Research Database (Denmark)

Bouzinova, Elena

Aim: Na,K-ATPase is essential for maintaining the transmembrane ion gradient and might initiate various intracellular signaling. These signals possibly act through a modification of the local ion concentrations or via Src-kinase activation. It is known that inhibition of the α-2 isoform of Na......,K-ATPase by ouabain elevates blood pressure. Consequently, ouabain was shown to potentiate arterial contraction in vitro. In contrast, we have demonstrated that siRNA-induced down-regulation of the α-2 isoform Na,K-ATPase expression reduced arterial sensitivity to agonist stimulation and prevented the effect......) phosphorylation assay. Down-regulation of the α-2 isoform Na,K-ATPase prevented the inhibitory effect of Src inhibitors on arterial contraction. Conclusions: The pro-contractile action of ouabain-sensitive Na,K-ATPase inhibition is associated with Src-kinase inhibition suggesting the role of this signaling...

A novel mechanism of P-type ATPase autoinhibition involving both termini of the protein

DEFF Research Database (Denmark)

Ekberg, Kira; Palmgren, Michael; Veierskov, Bjarke

2010-01-01

The activity of many P-type ATPases is found to be regulated by interacting proteins or autoinhibitory elements located in N- or C-terminal extensions. An extended C terminus of fungal and plant P-type plasma membrane H+-ATPases has long been recognized to be part of a regulatory apparatus....... This identifies the first group of P-type ATPases for which both ends of the polypeptide chain constitute regulatory domains, which together contribute to the autoinhibitory apparatus. This suggests an intricate mechanism of cis-regulation with both termini of the protein communicating to obtain the necessary...

New ATPase regulators-p97 goes to the PUB

DEFF Research Database (Denmark)

Madsen, Louise; Seeger, Michael; Semple, Colin A

2009-01-01

The conserved eukaryotic AAA-type ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast, is involved in a number of cellular pathways, including fusion of homotypic membranes, protein degradation, and activation of membrane-bound transcription factors. Most likely, p97 is directed...

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

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...

Pro-contractile action of the Na,K-ATPase/Src-kinase signaling pathway in the vascular wall

DEFF Research Database (Denmark)

Bouzinova, Elena; Aalkjær, Christian; Matchkov, Vladimir

Aim: Na,K-ATPase is essential for maintaining the transmembrane ion gradient and might initiate various intracellular signaling. These signals possibly act through a modification of the local ion concentrations or via Src-kinase activation. It is known that inhibition of the α-2 isoform of Na......,K-ATPase by ouabain elevates blood pressure. Consequently, ouabain was shown to potentiate arterial contraction in vitro. In contrast, we have demonstrated that siRNA-induced down-regulation of the α-2 isoform Na,K-ATPase expression reduced arterial sensitivity to agonist stimulation and prevented the effect......) phosphorylation assay. Down-regulation of the α-2 isoform Na,K-ATPase prevented the inhibitory effect of Src inhibitors on arterial contraction. Conclusions: The pro-contractile action of ouabain-sensitive Na,K-ATPase inhibition is associated with Src-kinase inhibition suggesting the role of this signaling...

Epithelial Na, K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

Directory of Open Access Journals (Sweden)

Martín-Vasallo Pablo

2003-06-01

Full Text Available Abstract Background An important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na+ gradients it generates are utilized for the Na+ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH and prostatic adenocarcinoma (PCa using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression. Results Expression of Na, K-ATPase α1 and β1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and BPH prostates. Canine kidney was used as positive control for expression of Na, K-ATPase α1 and γ isoforms. The α1 isoform was detected in abundance in prostatic epithelial cells but there was no evidence of α2, α3 or γ subunit expression. In advanced PCa, Na, K-ATPase α1 isoform expression was significantly lower compared to normal and BPH glands. The abundant basolateral immunostaining observed in normal and BPH tissue was significantly attenuated in PCa. Conclusion The loss of epithelial structure and function and the transformation of normal epithelial cells to malignant cells in the canine prostate have important implications for cellular metabolism and are accompanied by a down regulation of Na, K-ATPase.

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....

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.

Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres.

Science.gov (United States)

Avrova, Stanislava V; Rysev, Nikita A; Matusovsky, Oleg S; Shelud'ko, Nikolay S; Borovikov, Yurii S

2012-05-01

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin. Copyright © 2012 Elsevier Inc. All rights reserved.

Regulation of renal Na+-K-ATPase in the rat: role of increased potassium transport

International Nuclear Information System (INIS)

Mujais, S.K.; Chekal, M.A.; Hayslett, J.P.; Katz, A.I.

1986-01-01

The purpose of this study was to characterize the alterations in collecting tubule Na + -K + -ATPase activity produced by sustained increments in dietary potassium in the rat and to evaluate the role of aldosterone in their generation. In adrenal-intact animals, feeding a high-potassium diet or administration of a high physiological dose of aldosterone, which simulates the delivery rate of this hormone during potassium loading, caused marked increments in Na + -K + -ATPase activity in the cortical collecting tubule (CCT) but had no effect on the enzyme in the inner stripe of the medullary collecting tubule (MCT). A significant increase in enzyme activity was also observed after smaller dietary potassium increments and after 4 days of dietary potassium load. In adrenalectomized rats provided with physiological replacement doses of corticosterone and aldosterone, Na + -K + -ATPase activity in both CCT and MCT was similar to that of adrenal-intact controls but remained unchanged after 7 days on the potassium-enriched (10-fold) diet. In contrast, adrenalectomized animals receiving the high physiological dose of aldosterone displayed an increase in Na + -K + -ATPase activity of CCT comparable with that of adrenal-intact animals, whereas the enzyme activity in the MCT was unaffected. In conclusion, 1) following chronic potassium loading Na + -K + -ATPase activity increases significantly in the CCT with no change in its activity in the inner stripe of the MCT; 2) this increase in enzyme activity occurs in a time-dependent fashion and in proportion to the potassium load; and 3) the stimulation of Na + -K + -ATPase activity in adrenal-replaced rats is facilitated by augmented levels of aldosterone, such as those actually observed in adrenal-intact rats subjected to chronic potassium loading

Regulation of renal Na -K-ATPase in the rat: role of increased potassium transport

Energy Technology Data Exchange (ETDEWEB)

Mujais, S.K.; Chekal, M.A.; Hayslett, J.P.; Katz, A.I.

1986-08-01

The purpose of this study was to characterize the alterations in collecting tubule Na -K -ATPase activity produced by sustained increments in dietary potassium in the rat and to evaluate the role of aldosterone in their generation. In adrenal-intact animals, feeding a high-potassium diet or administration of a high physiological dose of aldosterone, which simulates the delivery rate of this hormone during potassium loading, caused marked increments in Na -K -ATPase activity in the cortical collecting tubule (CCT) but had no effect on the enzyme in the inner stripe of the medullary collecting tubule (MCT). A significant increase in enzyme activity was also observed after smaller dietary potassium increments and after 4 days of dietary potassium load. In adrenalectomized rats provided with physiological replacement doses of corticosterone and aldosterone, Na -K -ATPase activity in both CCT and MCT was similar to that of adrenal-intact controls but remained unchanged after 7 days on the potassium-enriched (10-fold) diet. In contrast, adrenalectomized animals receiving the high physiological dose of aldosterone displayed an increase in Na -K -ATPase activity of CCT comparable with that of adrenal-intact animals, whereas the enzyme activity in the MCT was unaffected. In conclusion, 1) following chronic potassium loading Na -K -ATPase activity increases significantly in the CCT with no change in its activity in the inner stripe of the MCT; 2) this increase in enzyme activity occurs in a time-dependent fashion and in proportion to the potassium load; and 3) the stimulation of Na -K -ATPase activity in adrenal-replaced rats is facilitated by augmented levels of aldosterone, such as those actually observed in adrenal-intact rats subjected to chronic potassium loading.

Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1.

Science.gov (United States)

Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg

2016-01-20

YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Our data identify a redox-regulated monomer-dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response.

Regulation of renal function and structure by the signaling Na/K-ATPase.

Science.gov (United States)

Xie, Jeffrey X; Li, Xin; Xie, Zijian

2013-12-01

The Na/K-ATPase as an essential ion pump was discovered more than 50 years ago (Skou (1989) Biochim. Biophys. Acta 1000, 439-446; Feraille and Doucet (2001) Physiol. Rev. 81, 345-418). The signaling function of Na/K-ATPase has been gradually appreciated over the last 20 years, first from the studies of regulatory effects of ouabain on cardiac cell growth. Several reviews on this topic have been written during the last few years (Schoner and Scheiner-Bobis (2007) Am. J. Physiol. Cell. Physiol. 293, C509-C536; Xie and Cai (2003) Mol. Interv. 3, 157 - 168; Bagrov et al. (2009) Pharmacol. Rev. 61, 9-38; Tian and Xie (2008) Physiology 23, 205-211; Fontana et al. (2013) FEBS J. 280, 5450-5455; Blanco and Wallace (2013) Am. J. Physiol. Renal Physiol. 305, F797-F812). This article will focus on the molecular mechanism of Na/K-ATPase-mediated signal transduction and its potential regulatory role in renal physiology and diseases. © 2013 International Union of Biochemistry and Molecular Biology.

Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio.

NARCIS (Netherlands)

Weigand, K.M.; Swarts, H.G.P.; Fedosova, N.U.; Russel, F.G.M.; Koenderink, J.B.

2012-01-01

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target

Protein phosphatase 2A interacts with the Na,K-ATPase and modulates its trafficking by inhibition of its association with arrestin.

Directory of Open Access Journals (Sweden)

Toru Kimura

Full Text Available The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na⁺,K⁺-ATPase. The catalytic subunit of the Na⁺,K⁺-ATPase includes several functional domains that determine its enzymatic and trafficking properties.Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A catalytic C-subunit is a specific Na⁺,K⁺-ATPase interacting protein. PP-2A C-subunit interacted with the Na⁺,K⁺-ATPase, but not with the homologous sequences of the H⁺,K⁺-ATPase. We confirmed that the Na⁺,K⁺-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs are important regulators of G-protein coupled receptor (GPCR signaling, and they also regulate Na⁺,K⁺-ATPase trafficking through direct association. PP2A inhibits association between the Na⁺,K⁺-ATPase and arrestin, and diminishes the effect of arrestin on Na⁺,K⁺-ATPase trafficking. GRK phosphorylates the Na⁺,K⁺-ATPase and PP2A can at least partially reverse this phosphorylation.Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase.

21-Benzylidene Digoxin: A Proapoptotic Cardenolide of Cancer Cells That Up-Regulates Na,K-ATPase and Epithelial Tight Junctions

Science.gov (United States)

Rocha, Sayonarah C.; Pessoa, Marco T. C.; Neves, Luiza D. R.; Alves, Silmara L. G.; Silva, Luciana M.; Santos, Herica L.; Oliveira, Soraya M. F.; Taranto, Alex G.; Comar, Moacyr; Gomes, Isabella V.; Santos, Fabio V.; Paixão, Natasha; Quintas, Luis E. M.; Noël, François; Pereira, Antonio F.; Tessis, Ana C. S. C.; Gomes, Natalia L. S.; Moreira, Otacilio C.; Rincon-Heredia, Ruth; Varotti, Fernando P.; Blanco, Gustavo; Villar, Jose A. F. P.; Contreras, Rubén G.; Barbosa, Leandro A.

2014-01-01

Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions. PMID:25290152

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...

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.

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.

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...

Hypoxia Stress Modifies Na+/K+-ATPase, H+/K+-ATPase, Na+/NH4+-ATPase, and nkaα1 Isoform Expression in the Brain of Immune-Challenged Air-Breathing Fish

Science.gov (United States)

Peter, MC Subhash; Simi, Satheesan

2017-01-01

Fishes are equipped to sense stressful stimuli and are able to respond to environmental stressor such as hypoxia with varying pattern of stress response. The functional attributes of brain to hypoxia stress in relation to ion transport and its interaction during immune challenge have not yet delineated in fish. We, therefore, explored the pattern of ion transporter functions and messenger RNA (mRNA) expression of α1-subunit isoforms of Na+/K+-ATPase (NKA) in the brain segments, namely, prosencephalon (PC), mesencephalon (MC), and metencephalon (MeC) in an obligate air-breathing fish exposed either to hypoxia stress (30 minutes forced immersion in water) or challenged with zymosan treatment (25-200 ng g−1 for 24 hours) or both. Zymosan that produced nonspecific immune responses evoked differential regulation of NKA, H+/K+-ATPase (HKA), and Na+/NH4+-ATPase (NNA) in the varied brain segments. On the contrary, hypoxia stress that demanded activation of NKA in PC and MeC showed a reversed NKA activity pattern in MeC of immune-challenged fish. A compromised HKA and NNA regulation during hypoxia stress was found in immune-challenged fish, indicating the role of these brain ion transporters to hypoxia stress and immune challenges. The differential mRNA expression of α1-subunit isoforms of NKA, nkaα1a, nkaα1b, and nkaα1c, in hypoxia-stressed brain showed a shift in its expression pattern during hypoxia stress-immune interaction in PC and MC. Evidence is thus presented for the first time that ion transporters such as HKA and NNA along with NKA act as functional brain markers which respond differentially to both hypoxia stress and immune challenges. Taken together, the data further provide evidence for a differential Na+, K+, H+, and NH4+ ion signaling that exists in brain neuronal clusters during hypoxia stress-immune interaction as a result of modified regulations of NKA, HKA, and NNA transporter functions and nkaα1 isoform regulation. PMID:29238219

Regulation of Na(+)/K(+)-ATPase by neuron-specific transcription factor Sp4: implication in the tight coupling of energy production, neuronal activity and energy consumption in neurons.

Science.gov (United States)

Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

2014-02-01

A major source of energy demand in neurons is the Na(+)/K(+)-ATPase pump that restores the ionic gradient across the plasma membrane subsequent to depolarizing neuronal activity. The energy comes primarily from mitochondrial oxidative metabolism, of which cytochrome c oxidase (COX) is a key enzyme. Recently, we found that all 13 subunits of COX are regulated by specificity (Sp) factors, and that the neuron-specific Sp4, but not Sp1 or Sp3, regulates the expression of key glutamatergic receptor subunits as well. The present study sought to test our hypothesis that Sp4 also regulates Na(+)/K(+)-ATPase subunit genes in neurons. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutational analysis, over-expression, and RNA interference studies, we found that Sp4, with minor contributions from Sp1 and Sp3, functionally regulate the Atp1a1, Atp1a3, and Atp1b1 subunit genes of Na(+)/K(+)-ATPase in neurons. Transcripts of all three genes were up-regulated by depolarizing KCl stimulation and down-regulated by the impulse blocker tetrodotoxin (TTX), indicating that their expression was activity-dependent. Silencing of Sp4 blocked the up-regulation of these genes induced by KCl, whereas over-expression of Sp4 rescued them from TTX-induced suppression. The effect of silencing or over-expressing Sp4 on primary neurons was much greater than those of Sp1 or Sp3. The binding sites of Sp factors on these genes are conserved among mice, rats and humans. Thus, Sp4 plays an important role in the transcriptional coupling of energy generation and energy consumption in neurons. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Mammary gland involution is associated with rapid down regulation of major mammary Ca**2+-ATPases

Science.gov (United States)

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca**2+-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca**2+-ATPases and mammary calcium transport is unknown. We found that 24 hours after stopping milk prod...

Quercetin treatment regulates the Na+,K+-ATPase activity, peripheral cholinergic enzymes, and oxidative stress in a rat model of demyelination.

Science.gov (United States)

Carvalho, Fabiano B; Gutierres, Jessié M; Beckmann, Diego; Santos, Rosmarini P; Thomé, Gustavo R; Baldissarelli, Jucimara; Stefanello, Naiara; Andrades, Amanda; Aiello, Graciane; Ripplinger, Angel; Lucio, Bruna M; Ineu, Rafael; Mazzanti, Alexandre; Morsch, Vera; Schetinger, Maria Rosa; Andrade, Cinthia M

2018-07-01

Quercetin is reported to exert a plethora of health benefits through many different mechanisms of action. This versatility and presence in the human diet has attracted the attention of the scientific community, resulting in a huge output of in vitro and in vivo (preclinical) studies. Therefore, we hypothesized that quercetin can protect Na + ,K + -ATPase activity in the central nervous system, reestablish the peripheral cholinesterases activities, and reduce oxidative stress during demyelination events in rats. In line with this expectation, our study aims to find out how quercetin acts on the Na + ,K + -ATPase activity in the central nervous system, peripheral cholinesterases, and stress oxidative markers in an experimental model of demyelinating disease. Wistar rats were divided into 4 groups: vehicle, quercetin, ethidium bromide (EB), and EB plus quercetin groups. The animals were treated once a day with vehicle (ethanol 20%) or quercetin 50 mg/kg for 7 (demyelination phase, by gavage) or 21 days (remyelination phase) after EB (0.1%, 10 μL) injection (intrapontine).The encephalon was removed, and the pons, hypothalamus, cerebral cortex, hippocampus, striatum, and cerebellum were dissected to verify the Na + ,K + -ATPase activity. Our results showed that quercetin protected against reduction in Na + ,K + -ATPase in the pons and cerebellum in the demyelination phase, and it increased the activity of this enzyme in the remyelination phase. During the demyelination, quercetin promoted the increase in acetylcholinesterase activity in whole blood and lymphocytes induced by EB, and it reduced the increase in acetylcholinesterase activity in lymphocytes in the remyelination phase. On day 7, EB increased the superoxide dismutase and decreased catalase activities, as well as increased the thiobarbituric acid-reactive substance levels. Taken together, these results indicated that quercetin regulates the Na + ,K + -ATPase activity, affects the alterations of redox state

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

Science.gov (United States)

Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc

2010-04-01

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens

Directory of Open Access Journals (Sweden)

Seema Mishra

2017-05-01

Full Text Available P1B-ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri. Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd2+ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn2+ and toxic Cd2+, but not at deficient Zn2+ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4. Further, in shoots NcHMA4 was more abundant in 10 μM Zn2+ and AhHMA4 in Zn2+ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn2+ when compared to replete Zn2+ and toxic Cd2+ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn2+ supply, but were up-regulated by 10 μM Cd2+. Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri, both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens, mesophyll in A. halleri.

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase

Directory of Open Access Journals (Sweden)

Isabelle Bouchez

2015-07-01

Full Text Available It has now been clearly shown that lipid droplets (LDs play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+ ATPase (V-ATPase. Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

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...

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.

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

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...

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....

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.

Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

Directory of Open Access Journals (Sweden)

Mi Young Yang

2013-05-01

Full Text Available GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

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.

Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K

Energy Technology Data Exchange (ETDEWEB)

Bowen, J.W.; McDonough, A.

1987-02-01

Long-term upregulation of the sodium pump (Na-K-adenosine triphosphatase (Na-K-ATPase)) entails an increase in the number of enzyme molecules. The authors incubated Madin-Darby canine kidney (MDCK) cells in low K medium and studied the time course and magnitude of change in the relative abundance of the two Na-K-ATPase subunits ( and US ), in the synthesis rate of the subunits, and in the relative abundance of - and US -mRNA. When cells were incubated in medium containing 0.25 mM K , intracellular Na increased. The relative abundance of Na-K-ATPase subunits, measured with ( SVI)-labelled immunoblots of cell homogenates, increases such that after 24 h was 1.71 +/- 0.33 and US was 1.67 +/- 0.22 times control. After 8 h of K depletion, -synthesis rate, measured by immunoprecipitation of pulse-labelled cells, increased to 2.30 +/- 0.50 and beta increased to 2.07 +/- 0.42 times control. The - and US -subunit mRNA abundance, measured by hybridizing - and US -cDNA probes to total RNA, increased within 30 min to 1.93 +/- 0.24 and 2.29 +/- 0.64 times control, respectively. They conclude that regulatory adjustments of Na-K-ATPase abundance involve an increase in translation after a rapid and coordinate increase in the concentrations of - and US -subunit mRNA.

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.

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

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.

Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

Science.gov (United States)

Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M; Haslett, Luke J; Kumar, Asok; Sato, Yutaka; Lie, Pearl P Y; Mohan, Panaiyur; Coffey, Erin E; Kompella, Uday; Mitchell, Claire H; Lloyd-Evans, Emyr; Nixon, Ralph A

2015-09-01

Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

Directory of Open Access Journals (Sweden)

Ju-Hyun Lee

2015-09-01

Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

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

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.

A Surfactant-Induced Functional Modulation of a Global Virulence Regulator from Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Sukhendu Mandal

Full Text Available Triton X-100 (TX-100, a useful non-ionic surfactant, reduced the methicillin resistance in Staphylococcus aureus significantly. Many S. aureus proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in S. aureus. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant S. aureus SarA (rSarA and its derivative (C9W have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future.

C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity.

Science.gov (United States)

Chalat, Madhavan; Moleschi, Kody; Molday, Robert S

2017-02-01

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2. © 2017 Chalat et al. 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).

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.

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.

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

DEFF Research Database (Denmark)

Cornelius, Flemming; Mahmmoud, Yasser Ahmed

2007-01-01

acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K...

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.

NO Metabolites Levels in Human Red Blood Cells are Affected by Palytoxin, an Inhibitor of Na(+)/K(+)-ATPase Pump.

Science.gov (United States)

Carelli-Alinovi, Cristiana; Tellone, Ester; Russo, Anna Maria; Ficarra, Silvana; Pirolli, Davide; Galtieri, Antonio; Giardina, Bruno; Misiti, Francesco

2014-01-01

Palytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulation.

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

RNAi-based silencing of genes encoding the vacuolar- ATPase ...

African Journals Online (AJOL)

RNAi-based silencing of genes encoding the vacuolar- ATPase subunits a and c in pink bollworm (Pectinophora gossypiella). Ahmed M. A. Mohammed. Abstract. RNA interference is a post- transcriptional gene regulation mechanism that is predominantly found in eukaryotic organisms. RNAi demonstrated a successful ...

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...

Molecular mechanism of bacterial Hsp90 pH-dependent ATPase activity.

Science.gov (United States)

Jin, Yi; Hoxie, Reyal S; Street, Timothy O

2017-06-01

Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated open-to-closed-to-open conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pH-dependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformation-specific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anti-correlation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255-ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255-ATP salt-bridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pH-dependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal site-specific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity. © 2017 The Protein Society.

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.

Nonspecific nature of the vanadate inhibition of rat ileal (Na, K)-ATPase

International Nuclear Information System (INIS)

Hajjar, J.J.; Rowe, W.A.; Tomicic, T.K.

1988-01-01

Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis the authors examined the stimulatory and inhibitory effects of vanadate on 86 Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal inhibition of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme

Nonspecific nature of the vanadate inhibition of rat ileal (Na, K)-ATPase

Energy Technology Data Exchange (ETDEWEB)

Hajjar, J.J.; Rowe, W.A.; Tomicic, T.K.

1988-01-01

Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis the authors examined the stimulatory and inhibitory effects of vanadate on /sup 86/Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal inhibition of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme.

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...

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

Endogenous sodium potassium ATPase inhibition related biochemical cascade and the acquired immunodeficiency syndrome -Neural regulation of viral replication and immune response to the virus

Directory of Open Access Journals (Sweden)

Ravikumar A

2001-11-01

Full Text Available The isoprenoid pathway and its metabolites - digoxin, dolichol and ubiquinone were assessed in acquired immunodeficiency syndrome. Digoxin is an endogenous regulator of membrane Na+-K+ ATPase secreted by the human hypothalamus. The HMG CoA reductase activity was increased with increased digoxin and dolichol levels and reduced ubiquinone levels in AIDS. Membrane Na+-K+ ATPase activity and serum magnesium levels were reduced. The tryptophan catabolites were increased and the tyrosine catabolites were reduced. The glycoconjugate metabolites were increased and lysosomal stability was reduced. There was reduced incorporation of glycoconjugates into membranes and increased membrane cholesterol: phospholipid ratio. Lipid peroxidation products and NO were increased while free radical scavenging enzymes and reduced glutathione were reduced. The role of the isoprenoid pathway related cascade in the pathogenesis of AIDS is discussed.

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.

Hypoxia Stress Modifies Na/K-ATPase, H/K-ATPase, , and Isoform Expression in the Brain of Immune-Challenged Air-Breathing Fish

Directory of Open Access Journals (Sweden)

MC Subhash Peter

2017-11-01

Full Text Available Fishes are equipped to sense stressful stimuli and are able to respond to environmental stressor such as hypoxia with varying pattern of stress response. The functional attributes of brain to hypoxia stress in relation to ion transport and its interaction during immune challenge have not yet delineated in fish. We, therefore, explored the pattern of ion transporter functions and messenger RNA (mRNA expression of α1-subunit isoforms of Na + /K + -ATPase (NKA in the brain segments, namely, prosencephalon (PC, mesencephalon (MC, and metencephalon (MeC in an obligate air-breathing fish exposed either to hypoxia stress (30 minutes forced immersion in water or challenged with zymosan treatment (25-200 ng g −1 for 24 hours or both. Zymosan that produced nonspecific immune responses evoked differential regulation of NKA, H + /K + -ATPase (HKA, and Na + / NH 4 + - ATPase (NNA in the varied brain segments. On the contrary, hypoxia stress that demanded activation of NKA in PC and MeC showed a reversed NKA activity pattern in MeC of immune-challenged fish. A compromised HKA and NNA regulation during hypoxia stress was found in immune-challenged fish, indicating the role of these brain ion transporters to hypoxia stress and immune challenges. The differential mRNA expression of α1-subunit isoforms of NKA, nkaα1a , nkaα1b , and nkaα1c , in hypoxia-stressed brain showed a shift in its expression pattern during hypoxia stress-immune interaction in PC and MC. Evidence is thus presented for the first time that ion transporters such as HKA and NNA along with NKA act as functional brain markers which respond differentially to both hypoxia stress and immune challenges. Taken together, the data further provide evidence for a differential Na + , K + , H + , and NH 4 + ion signaling that exists in brain neuronal clusters during hypoxia stress-immune interaction as a result of modified regulations of NKA, HKA, and NNA transporter functions and nkaα1 isoform

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.

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.

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.

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.

Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E(2)P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump.

Science.gov (United States)

Mahmmoud, Yasser A; Christensen, S Brøgger

2011-10-01

Nigella sativa seed oil was found to contain a modulator of Na,K-ATPase. Separation analyses combined with (1)H NMR and GCMS identified the inhibitory fraction as a mixture of oleic and linoleic acids. These two fatty acids are specifically concentrated in several medicinal plant oils, and have particularly been implicated in decreasing high blood pressure. The ouabain binding site on Na,K-ATPase has also been implicated in blood pressure regulation. Thus, we aimed to determine how these two molecules modify pig kidney Na,K-ATPase. Oleic and linoleic acids did not modify reactions involving the E(1) (Na(+)) conformations of the Na,K-ATPase. In contrast, K(+) dependent reactions were strongly modified after treatment. Oleic and linoleic acids were found to stabilize a pump conformation that binds ouabain with high affinity, i.e., an ion free E(2)P form. Time-resolved binding assays using anthroylouabain, a fluorescent ouabain analog, revealed that the increased ouabain affinity is unique to oleic and linoleic acids, as compared with γ-linolenic acid, which decreased pump-mediated ATP hydrolysis but did not equally increase ouabain interaction with the pump. Thus, the dynamic changes in plasma levels of oleic and linoleic acids are important in the modulation of the sensitivity of the sodium pump to cardiac glycosides. Given the possible involvement of the cardiac glycoside binding site on Na,K-ATPase in the regulation of hypertension, we suggest oleic acid to be a specific chaperon that modulates interaction of cardiac glycosides with the sodium pump. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Maintained inspiratory activity during proportional assist ventilation in surfactant-depleted cats early after surfactant instillation: phrenic nerve and pulmonary stretch receptor activity

Directory of Open Access Journals (Sweden)

Schaller Peter

2006-03-01

Full Text Available Abstract Background Inspiratory activity is a prerequisite for successful application of patient triggered ventilation such as proportional assist ventilation (PAV. It has recently been reported that surfactant instillation increases the activity of slowly adapting pulmonary stretch receptors (PSRs followed by a shorter inspiratory time (Sindelar et al, J Appl Physiol, 2005 [Epub ahead of print]. Changes in lung mechanics, as observed in preterm infants with respiratory distress syndrome and after surfactant treatment, might therefore influence the inspiratory activity when applying PAV early after surfactant treatment. Objective To investigate the regulation of breathing and ventilatory response in surfactant-depleted young cats during PAV and during continuous positive airway pressure (CPAP early after surfactant instillation in relation to phrenic nerve activity (PNA and the activity of PSRs. Methods Seven anesthetized, endotracheally intubated young cats were exposed to periods of CPAP and PAV with the same end-expiratory pressure (0.2–0.5 kPa before and after lung lavage and after surfactant instillation. PAV was set to compensate for 75% of the lung elastic recoil. Results Tidal volume and respiratory rate were higher with lower PaCO2 and higher PaO2 during PAV than during CPAP both before and after surfactant instillation (p Conclusion PSR activity and the control of breathing are maintained during PAV in surfactant-depleted cats early after surfactant instillation, with a higher ventilatory response and a lower breathing effort than during CPAP.

Isolation and characterization of a specific endogenous Na+, K+-ATPase inhibitor from bovine adrenal

International Nuclear Information System (INIS)

Tamura, M.; Lam, T.T.; Inagami, T.

1988-01-01

In order to identify a specific endogenous Na + ,K + -ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na + ,K + -ATPase; (ii) competitive displacing activity against [ 3 H]ouabain binding to the enzyme; (iii) inhibitory activity for 86 Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C 18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na + ,K + -ATPase. These results strongly suggest that this water-soluble nonpeptidic Na + ,K + -ATPase inhibitor may be a specific endogenous regulator for the ATPase

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

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.

Mutation of the Na+/K+-ATPase Atp1a1a.1 causes QT interval prolongation and bradycardia in zebrafish.

Science.gov (United States)

Pott, Alexander; Bock, Sarah; Berger, Ina M; Frese, Karen; Dahme, Tillman; Keßler, Mirjam; Rinné, Susanne; Decher, Niels; Just, Steffen; Rottbauer, Wolfgang

2018-05-08

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na + /K + -ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na + /K + -ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na + /K + -ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na + /K + -ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na + /K + -ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization. Copyright © 2018. Published by Elsevier Ltd.

MICROBIAL SURFACTANTS. I. GLYCOLIPIDS

Directory of Open Access Journals (Sweden)

Pirog T. Р.

2014-02-01

Full Text Available The review is devoted to surface-active glycolipids. The general characteristics, the physiological role of the rhamnolipids, trehalose lipids, sophorolipids, mannosylerythritol lipids and their traditional producers — the representatives of the genera Pseudozyma, Pseudomonas, Rhodococcus and Candida are given. The detailed analysis of the chemical structure, the stages of the biosynthesis and the regulation of some low molecular glycolipids are done. The own experimental data concerning the synthesis intensification, the physiological role and the practical use of Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 surfactants, which are a complex of the glyco-, phospho-, amino- and neutral lipids (glycolipids of all strains are presented by trehalose mycolates are summarized. It was found that R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 surfactants have protective, antimicrobial and antiadhesive properties. It was shown that R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 surfactants preparation of cultural liquid intensified the degradation of oil in water due to the activation of the natural petroleum-oxidizing microflora.

Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU.

Science.gov (United States)

Silberg, Jonathan J; Tapley, Tim L; Hoff, Kevin G; Vickery, Larry E

2004-12-24

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.

Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase.

Science.gov (United States)

Li, Ciming; Crambert, Gilles; Thuillard, Delphine; Roy, Sophie; Schaer, Danièle; Geering, Käthi

2005-12-30

Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,K-ATPase. Mutational effects on the stable association with Na,K-ATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. Ile-35, Ile-43, and Ile-44 are only involved in the stable association with Na,K-ATPase. Glu-26, Met-30, and Ile-44 are important for the functional effect and/or the efficient association of FXYD7 with Na,K-ATPase, consistent with the prediction that these amino acids contact TM domain 9 of the alpha subunit (Li, C., Grosdidier, A., Crambert, G., Horisberger, J.-D., Michielin, O., and Geering, K. (2004) J. Biol. Chem. 279, 38895-38902). Several amino acids that are not implicated in the efficient association of FXYD7 with the Na,K-ATPase are specifically involved in the functional effect of FXYD7. Leu-32 and Phe-37 influence the apparent affinity for external K+, whereas Val-28 and Ile-42 are implicated in the apparent affinity for both external K+ and external Na+. These amino acids act in a synergistic way. These results highlight the important structural and functional role of the TM domain of FXYD7 and delineate the determinants that mediate the complex interactions of FXYD7 with Na,K-ATPase.

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

Science.gov (United States)

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.

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.

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...

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.

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.

Plasma membrane calcium ATPases and related disorders.

Science.gov (United States)

Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

2013-03-01

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of a mutant α1 Na/K-ATPase that pumps but is defective in signal transduction.

Science.gov (United States)

Lai, Fangfang; Madan, Namrata; Ye, Qiqi; Duan, Qiming; Li, Zhichuan; Wang, Shaomeng; Si, Shuyi; Xie, Zijian

2013-05-10

It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. A420P mutant has normal pumping but not signaling function. Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.

Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

Directory of Open Access Journals (Sweden)

Ahmad Almilaji

2013-05-01

Full Text Available Background/Aims: Human parvovirus B19 (B19V may cause inflammatory cardiomyopathy (iCMP which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A and K+ induced pump current (Ipump as well as ouabain-inhibited current (Iouabain both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml. According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml similarly decreased Ipump in human microvascular endothelial cells (HMEC. Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2 dependent formation of lysophosphatidylcholine.

Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy

Directory of Open Access Journals (Sweden)

Bondar Alexander

2011-01-01

Full Text Available Abstract Background The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform in the postsynaptic region of the spine. Conclusions A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

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

Surfactant phosphatidylcholine metabolism and surfactant function in preterm, ventilated lambs

International Nuclear Information System (INIS)

Jobe, A.H.; Ikegami, M.; Seidner, S.R.; Pettenazzo, A.; Ruffini, L.

1989-01-01

Preterm lambs were delivered at 138 days gestational age and ventilated for periods up to 24 h in order to study surfactant metabolism and surfactant function. The surfactant-saturated phosphatidylcholine pool in the alveolar wash was 13 +/- 4 mumol/kg and did not change from 10 min to 24 h after birth. Trace amounts of labeled natural sheep surfactant were mixed with fetal lung fluid at birth. By 24 h, 80% of the label had become lung-tissue-associated, yet there was no loss of label from phosphatidylcholine in the lungs when calculated as the sum of the lung tissue plus alveolar wash. De novo synthesized phosphatidylcholine was labeled with choline given by intravascular injection at 1 h of age. Labeled phosphatidylcholine accumulated in the lung tissue linearly to 24 h, and the labeled phosphatidylcholine moved through lamellar body to alveolar pools. The turnover time for alveolar phosphatidylcholine was estimated to be about 13 h, indicating an active metabolic pool. A less surface-active surfactant fraction recovered as a supernatant after centrifugation of the alveolar washes at 40,000 x g increased from birth to 10 min of ventilation, but no subsequent changes in the distribution of surfactant phosphatidylcholine in surfactant fractions occurred. The results were consistent with recycling pathway(s) that maintained surface-active surfactant pools in preterm ventilated lambs

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.

Persurf, a new method to improve surfactant delivery: a study in surfactant depleted rats.

Directory of Open Access Journals (Sweden)

Wolfram Burkhardt

Full Text Available PURPOSE: Exogenous surfactant is not very effective in adults with ARDS, since surfactant does not reach atelectatic alveoli. Perfluorocarbons (PFC can recruit atelectatic areas but do not replace impaired endogenous surfactant. A surfactant-PFC-mixture could combine benefits of both therapies. The aim of the proof-of-principal-study was to produce a PFC-in-surfactant emulsion (Persurf and to test in surfactant depleted Wistar rats whether Persurf achieves I. a more homogenous pulmonary distribution and II. a more homogenous recruitment of alveoli when compared with surfactant or PFC alone. METHODS: Three different PFC were mixed with surfactant and phospholipid concentration in the emulsion was measured. After surfactant depletion, animals either received 30 ml/kg of PF5080, 100 mg/kg of stained (green dye Curosurf™ or 30 ml/kg of Persurf. Lungs were fixated after 1 hour of ventilation and alveolar aeration and surfactant distribution was estimated by a stereological approach. RESULTS: Persurf contained 3 mg/ml phospholipids and was stable for more than 48 hours. Persurf-administration improved oxygenation. Histological evaluation revealed a more homogenous surfactant distribution and alveolar inflation when compared with surfactant treated animals. CONCLUSIONS: In surfactant depleted rats administration of PFC-in-surfactant emulsion leads to a more homogenous distribution and aeration of the lung than surfactant alone.

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

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.

Gill ATPase activity in Procambarus clarkii as an indicator of heavy metal pollution

Energy Technology Data Exchange (ETDEWEB)

Torreblanca, A.; Del Ramo, J.; Diaz-Mayans, J. (Univ. of Valencia (Spain))

1989-06-01

Lake Albufera and the surrounding rice field waters are subjected to very heavy loads of sewage and toxic industrial residues, including heavy metals, from the many urban and waste waters of this area. The American red crayfish, Procambarus clarkii have a high resistance to toxic effects of heavy metals. The sublethal effects of heavy metals on gills of fish and aquatic invertebrates have been extensively studied. Some metabolic disturbances and histologic damages have been reported, as well as osmoregulation alterations. However, little work has been done about the effect of heavy metals on Na,K and Mg-ATPases of freshwater invertebrate gills. Na,K-ATPase is the prime mediator of ion transport across cellular membranes and plays a central role in whole body ion regulation in marine and estuarine animals. Na,K-ATPase has been reviewed and assessed as a potentially useful indicator of pollution stress in aquatic animals. The purpose of this study is look for the relation, if any, between crayfish gill ATP-ase activity changes and metal exposure in laboratory. This find would allow the authors to assay this potential indicator in the field.

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

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

Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

Energy Technology Data Exchange (ETDEWEB)

Tamura, M.; Lam, T.T.; Inagami, T.

1988-06-14

In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

[Determination of contact angle of pharmaceutical excipients and regulating effect of surfactants on their wettability].

Science.gov (United States)

Hua, Dong-dong; Li, He-ran; Yang, Bai-xue; Song, Li-na; Liu, Tiao-tiao; Cong, Yu-tang; Li, San-ming

2015-10-01

To study the effects of surfactants on wettability of excipients, the contact angles of six types of surfactants on the surface of two common excipients and mixture of three surfactants with excipients were measured using hypsometry method. The results demonstrated that contact angle of water on the surface of excipients was associated with hydrophilcity of excipients. Contact angle was lowered with increase in hydrophilic groups of excipient molecules. The sequence of contact angle from small to large was starch contact angle of excipients, and their abilities to lower contact angle varied. The results of the present study offer a guideline in the formulation design of tablets.

Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging.

Science.gov (United States)

Bernhem, Kristoffer; Blom, Hans; Brismar, Hjalmar

2018-01-01

Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Directory of Open Access Journals (Sweden)

I. Stefanon

2009-10-01

Full Text Available Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group underwent left coronary artery ligation (infarcted, Inf or sham-operation (Sham. Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05. The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05, while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity

DEFF Research Database (Denmark)

Martuseviciene, Giedre; Hofman-Bang, Jacob; Clausen, Torben

2011-01-01

increased in response to ethylene glycol tetraacetic acid-induced acute hypocalcemia and to the same extent in both vehicle and ouabain groups. The glands were removed, and inhibition of the ATPase was measured by (86)rubidium uptake, which was found to be significantly decreased in ouabain......-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect...... on the secretory response to acute hypocalcemia. Hence, the suggested importance of this ATPase in the regulation of PTH secretion could not be confirmed in this in vivo model....

Effect of fullerene C(60 on ATPase activity and superprecipitation of skeletal muscle actomyosin

Directory of Open Access Journals (Sweden)

K. S. Andreichenko

2013-04-01

Full Text Available Creation of new biocompatible nanomaterials, which can exhibit the specific biological effects, is an important complex problem that requires the use of last accomplishments of biotechnology. The effect of pristine water-soluble fullerene C60 on ATPase activity and superprecipitation reaction of rabbit skeletal muscle natural actomyosin has been revealed, namely an increase of actomyosin superprecipitation and Мg2+, Са2+– and K+-ATPase activity by fullerene was investigated. We conclude that this finding offers a real possibility for the regulation of contraction-relaxation of skeletal muscle with fullerene C60.

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

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

Science.gov (United States)

Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

ATPase and GTPase Tangos Drive Intracellular Protein Transport.

Science.gov (United States)

Shan, Shu-Ou

2016-12-01

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Electrostatic Stabilization Plays a Central Role in Autoinhibitory Regulation of the Na+,K+-ATPase

DEFF Research Database (Denmark)

Jiang, Qiucen; Garcia, Alvaro; Han, Minwoo

2017-01-01

The Na+,K+-ATPase is present in the plasma membrane of all animal cells. It plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane, which are essential in numerous physiological processes, e.g., nerve, muscle, and kidney function. Its cellular a...

Ouabain stimulates a Na+/K+-ATPase-mediated SFK-activated signalling pathway that regulates tight junction function in the mouse blastocyst.

Directory of Open Access Journals (Sweden)

Holly Giannatselis

Full Text Available The Na(+/K(+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na(+/K(+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10(-3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10(-4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.

Altered Regulation of type 3 Na+/H+ exchanger, type 1 Na+/HCO3- cotransporter, and Na+,K+-ATPase in the Kidney of Rats with Experimental Rhabdomyolysis

Science.gov (United States)

Ma, Seong Kwon; Bae, Eun Hui; Lee, JongUn; Kim, Sun Young; Kim, Sung Zoo; Choi, Ki Chul

2007-01-01

Metabolic acidosis was shown to correlate with deterioration of renal function in patients with rhabdomyolysis. The present study was aimed to investigate whether the changes of type 3 Na+/H+ exchanger (NHE3), type 1 Na+/HCO3- cotransporter (NBC1), and Na+,K+-ATPase α1 subunit may play a role in the pathogenesis of metabolic acidosis in glycerol-induced experimental rhabdomyolysis. Male Sprague-Dawley rats were deprived of fluid intake for 24 hours, and then were injected with 50% glycerol in normal saline (10 mL/kg, intramuscularly). At 24 hours after the glycerol injection, rats were sacrificed by decapitation. Control rats were injected with normal saline. The protein expression of NHE3, NBC1 and Na+,K+-ATPase α1 subunit was determined in the cortex of the kidney by immunoblotting and immunohistochemistry. Following the treatment of glycerol, creatinine clearance was significantly decreased, and high anion gap metabolic acidosis developed. In the experimental group, the expression of Na+,K+-ATPase α1 subunit was significantly decreased in the cortex of the kidney. On the contrary, the expression of NHE3 and NBC1 was significantly increased. Immunohistochemical analyses confirmed the immunoblotting data. In conclusion, the coordinate up-regulation of NHE3 and NBC1 may play an adaptive role against the metabolic acidosis in glycerol-induced rhabdomyolysis. PMID:24459502

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.

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...

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,...

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

Science.gov (United States)

Jakubowska, Dagmara; Janicka, Małgorzata

2017-11-01

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H + -ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H + -ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H + -ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H + -ATPase and NADPH oxidase are key factors. Copyright © 2017 Elsevier B.V. All rights reserved.

Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

Directory of Open Access Journals (Sweden)

Brian Roland eLarsen

2016-04-01

Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

Science.gov (United States)

Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

2018-01-01

It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

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

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

Science.gov (United States)

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.

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

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.

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.

Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase

DEFF Research Database (Denmark)

Poulsen, Hanne; Khandelia, Himanshu; Morth, J Preben

2010-01-01

severe neurological diseases. This novel model for ion transport by the Na(+)/K(+)-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely...

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

Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

Science.gov (United States)

Mahmmoud, Yasser A; Shattock, Michael; Cornelius, Flemming; Pavlovic, Davor

2014-01-01

Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

Pulmonary surfactant and lung transplantation

NARCIS (Netherlands)

Erasmus, Michiel Elardus

1997-01-01

Pulmonary surfactant lowers the surface tension at the air-water interface inside the alveolus. This is achieved by adsorption of surfactant phospholipids at the air-water interface, a process controlled by surfactant-associated proteins, such as SP-A. In this way, surfactant prevents collapse of

Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

International Nuclear Information System (INIS)

Dong, C.-Z.; Montillet, J.-L.; Triantaphylides, C.

1994-01-01

The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells (Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H + -ATPase activity. Plasma membrane-enriched vesicles were prepared and the H + -ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H + -ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2mM dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H + -ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH. This rapid irreversible inhibition of the plasma membrane H + -ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material. (author)

Na+K+-ATPase activity and K+ channels differently contribute to vascular relaxation in male and female rats.

Directory of Open Access Journals (Sweden)

Fernanda Moura Vargas Dias

Full Text Available Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones' effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K+ channels and Na+K+-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K+ channels and Na+K+-ATPase in vascular reactivity. Acetylcholine (ACh-induced relaxation was analyzed in the presence of L-NAME (100 µM and the following K+ channels blockers: tetraethylammonium (TEA, 2 mM, 4-aminopyridine (4-AP, 5 mM, iberiotoxin (IbTX, 30 nM, apamin (0.5 µM and charybdotoxin (ChTX, 0.1 µM. The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K+ channel (Kv participates more in males. Inhibition of the three types of Ca2+-activated K+ channels induced a greater reduction in Rmax in females than in males. The functional activity of the Na+K+-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUA incubation. OUA reduced K+-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na+K+-ATPase functional activity. L-NAME reduced K+-induced relaxation only in the female group, suggesting that nitric oxide (NO participates more in their functional Na+K+-ATPase activity. These results suggest that the K+ channels involved in the gender-based vascular relaxation differences are the large conductance Ca2+-activated K+ channels (BKCa in females and Kv in males and in the K+-induced relaxation and the Na+K+-ATPase vascular functional activity is greater in males.

Iron oxide nanoparticles to an Indian major carp, Labeo rohita: Impacts on hematology, iono regulation and gill Na+/K+ ATPase activity

Directory of Open Access Journals (Sweden)

Anand Sadanandan Remya

2015-04-01

Full Text Available In this study, the chronic toxicity effects of iron oxide (Fe2O3 nanoparticles (NPs (500 mg l−l on certain hematological, ionoregulatory and gill Na+/K+ ATPase activity of an Indian major carp, Labeo rohita were estimated for a period of 25 days under static bioassay. A significant increase in hemoglobin (Hb content, red blood cell (RBC count and hematocrit (Ht value was noticed throughout the study period when compared to control groups. In contrast, mean cellular volume (MCV, mean cellular hemoglobin (MCH (except on 5th day and mean cellular hemoglobin concentration (MCHC levels and white blood cell (WBC counts were found to be decreased during the above study period. Fe2O3 NPs also caused alterations in iono regulation resulting in hyponatremia (Na+, hypochloremia (Cl− (except on 5th day and hypokalemia (K+ (except up to 15th day. A biphasic trend in gill Na+/K+-ATPase activity was noticed during the above treatment period. Our results demonstrate that high Fe2O3 NP concentrations in the aquatic environment may have adverse physiological effects on fish. These data may be useful to assess the environmental risk posed by NPs. However the toxicity of various sizes of the nanoparticle could be evaluated using different aquatic organisms.

Microemulsion-based lycopene extraction: Effect of surfactants, co-surfactants and pretreatments.

Science.gov (United States)

Amiri-Rigi, Atefeh; Abbasi, Soleiman

2016-04-15

Lycopene is a potent antioxidant that has received extensive attention recently. Due to the challenges encountered with current methods of lycopene extraction using hazardous solvents, industry calls for a greener, safer and more efficient process. The main purpose of present study was application of microemulsion technique to extract lycopene from tomato pomace. In this respect, the effect of eight different surfactants, four different co-surfactants, and ultrasound and enzyme pretreatments on lycopene extraction efficiency was examined. Experimental results revealed that application of combined ultrasound and enzyme pretreatments, saponin as a natural surfactant, and glycerol as a co-surfactant, in the bicontinuous region of microemulsion was the optimal experimental conditions resulting in a microemulsion containing 409.68±0.68 μg/glycopene. The high lycopene concentration achieved, indicates that microemulsion technique, using a low-cost natural surfactant could be promising for a simple and safe separation of lycopene from tomato pomace and possibly from tomato industrial wastes. Copyright © 2015 Elsevier Ltd. All rights reserved.

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

Science.gov (United States)

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

2018-01-26

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

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

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

Science.gov (United States)

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

Effects of electric fields on membrane-bound Na, K-ATPase. Progress report, 1 July 1989-30 June 1990

Energy Technology Data Exchange (ETDEWEB)

Tsong, T.Y.

1990-06-30

We continued to work on effects of oscillating electric fields on membrane functions, in particular the electric activation of Na, K-ATPase, and to develop theory of electro-conformational coupling. We believe transmembrane electric fields are involved in the regulation of the internal activity of a cell and also in the cell-to-cell communications. An in depth study of Na, K-ATPase will provide useful information concerning the molecular design of a cell to sense and to transmit signals.

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.

Structure and Conformational Dynamics of DMPC/Dicationic Surfactant and DMPC/Dicationic Surfactant/DNA Systems

Directory of Open Access Journals (Sweden)

Maciej Kozak

2013-04-01

Full Text Available Amphiphilic dicationic surfactants, known as gemini surfactants, are currently studied for gene delivery purposes. The gemini surfactant molecule is composed of two hydrophilic “head” groups attached to hydrophobic chains and connected via molecular linker between them. The influence of different concentrations of 1,5-bis (1-imidazolilo-3-decyloxymethyl pentane chloride (gemini surfactant on the thermotropic phase behaviour of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC bilayers with and without the presence of DNA was investigated using Fourier transformed infrared (FTIR and circular dichroism (CD spectroscopies, small angle scattering of synchrotron radiation and differential scanning calorimetry. With increasing concentration of surfactant in DMPC/DNA systems, a disappearance of pretransition and a decrease in the main phase transition enthalpy and temperature were observed. The increasing intensity of diffraction peaks as a function of surfactant concentration also clearly shows the ability of the surfactant to promote the organisation of lipid bilayers in the multilayer lamellar phase.

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.

Surfactant Membrane Phases Containing Mixtures of Hydrocarbon and Fluorocarbon Surfactants

International Nuclear Information System (INIS)

de Campo, Liliana; Warr, G.G.

2005-01-01

Full text: We describe the structure and stability of sponge and lamellar phases comprising mixtures of hydrocarbon and fluorocarbon surfactants. Such mixtures can show limited miscibility with each other, forming for example coexisting populations of hydrocarbon rich and fluorocarbon rich micelles under some circumstances. Our system is based on the well-characterised lamellar and sponge phases of cetylpyridinium chloride, hexanol and 0.2M brine, into which the partially fluorinated surfactant N-1H,1H,2H,2H-tridecafluorooctylpyridinium chloride is incorporated. By probing the structures with SAXS (small angle x-ray scattering) and SANS (small angle neutron scattering) using contrast variation, and by characterizing the dynamic properties with dynamic light scattering, we will describe the effect of incorporating the fluorinated surfactant on the phase equilibria and properties of the surfactant membrane structures. (authors)

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

Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

Science.gov (United States)

Schewe, Bettina; Schmälzlin, Elmar; Walz, Bernd

2008-03-01

Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: implications for membrane-water interfacial arginines.

Science.gov (United States)

Gong, Xiao-Min; Ding, Yi; Yu, Jinghua; Yao, Yong; Marassi, Francesca M

2015-01-01

FXYD2 is a membrane protein responsible for regulating the function of the Na,K-ATPase in mammalian kidney epithelial cells. Here we report the structure of FXYD2b, one of two splice variants of the protein, determined by NMR spectroscopy in detergent micelles. Solid-state NMR characterization of the protein embedded in phospholipid bilayers indicates that several arginine side chains may be involved in hydrogen bond interactions with the phospholipid polar head groups. The structure and the NMR data suggest that FXYD2b could regulate the Na,K-ATPase by modulating the effective membrane surface electrostatics near the ion binding sites of the pump. Copyright © 2014 Elsevier B.V. All rights reserved.

Surfactant-Mediated Growth Revisited

International Nuclear Information System (INIS)

Meyerheim, H. L.; Sander, D.; Popescu, R.; Pan, W.; Kirschner, J.; Popa, I.

2007-01-01

The x-ray structure analysis of the oxygen-surfactant-mediated growth of Ni on Cu(001) identifies up to 0.15 monolayers of oxygen in subsurface octahedral sites. This questions the validity of the general view that surfactant oxygen floats on top of the growing Ni film. Rather, the surfactant action is ascribed to an oxygen-enriched zone extending over the two topmost layers. Surface stress measurements support this finding. Our results have important implications for the microscopic understanding of surfactant-mediated growth and the change of the magnetic anisotropy of the Ni films

Surfactants in tribology

CERN Document Server

Biresaw, Girma

2014-01-01

Surface science and tribology play very critical roles in many industries. Manufacture and use of almost all consumer and industrial products rely on the application of advanced surface and tribological knowledge. The fourth in a series, Surfactants in Tribology, Volume 4 provides an update on research and development activities connecting surfactants and tribological phenomena. Written by renowned subject matter experts, the book demonstrates how improved design of surfactants can be harnessed to control tribological phenomena. Profusely illustrated and copiously referenced, the chapters also

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

Science.gov (United States)

Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-04-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

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

Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

Directory of Open Access Journals (Sweden)

Young Ou

Full Text Available Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.

Plasma membrane H(+)-ATPase is involved in methyl jasmonate-induced root hair formation in lettuce (Lactuca sativa L.) seedlings.

Science.gov (United States)

Zhu, Changhua; Yang, Na; Ma, Xiaoling; Li, Guijun; Qian, Meng; Ng, Denny; Xia, Kai; Gan, Lijun

2015-06-01

Our results show that methyl jasmonate induces plasma membrane H (+) -ATPase activity and subsequently influences the apoplastic pH of trichoblasts to maintain a cell wall pH environment appropriate for root hair development. Root hairs, which arise from root epidermal cells, are tubular structures that increase the efficiency of water absorption and nutrient uptake. Plant hormones are critical regulators of root hair development. In this study, we investigated the regulatory role of the plasma membrane (PM) H(+)-ATPase in methyl jasmonate (MeJA)-induced root hair formation. We found that MeJA had a pronounced effect on the promotion of root hair formation in lettuce seedlings, but that this effect was blocked by the PM H(+)-ATPase inhibitor vanadate. Furthermore, MeJA treatment increased PM H(+)-ATPase activity in parallel with H(+) efflux from the root tips of lettuce seedlings and rhizosphere acidification. Our results also showed that MeJA-induced root hair formation was accompanied by hydrogen peroxide accumulation. The apoplastic acidification acted in concert with reactive oxygen species to modulate root hair formation. Our results suggest that the effect of MeJA on root hair formation is mediated by modulation of PM H(+)-ATPase activity.

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

Surfactant replacement therapy--economic impact.

Science.gov (United States)

Pejaver, R K; al Hifzi, I; Aldussari, S

2001-06-01

Surfactant replacement is an effective treatment for neonatal respiratory distress syndrome. (RDS). As widespread use of surfactant is becoming a reality, it is important to assess the economic implications of this new form of therapy. A comparison study was carried out at the Neonatal Intensive Care Unit (NICU) of Northwest Armed Forces Hospital, Saudi Arabia. Among 75 infants who received surfactant for RDS and similar number who were managed during time period just before the surfactant was available, but by set criteria would have made them eligible for surfactant. All other management modalities except surfactant were the same for all these babies. Based on the intensity of monitoring and nursing care required by the baby, the level of care was divided as: Level IIIA, IIIB, Level II, Level I. The cost per day per bed for each level was calculated, taking into account the use of hospital immovable equipment, personal salaries of nursing, medical, ancillary staff, overheads and maintenance, depreciation and replacement costs. Medications used, procedures done, TPN, oxygen, were all added to individual patient's total expenditure. 75 infants in the Surfactant group had 62 survivors. They spent a total of 4300 days in hospital. (av 69.35) Out of which 970 d (av 15.65 per patient) were ventilated days. There were 56 survivors in the non-surfactant group of 75. They had spent a total of 5023 days in the hospital (av 89.69/patient) out of which 1490 were ventilated days (av 26.60 d). Including the cost of surfactant (two doses), cost of hospital stay for each infant taking the average figures of stay would be SR 118, 009.75 per surfactant treated baby and SR 164, 070.70 per non-surfactant treated baby. The difference of 46,061 SR is 39.03% more in non-surfactant group. One Saudi rial = 8 Rs (approx at the time study was carried out.) Medical care cost varies from place to place. However, it is definitely cost-effective where surfactant is concerned. Quality adjusted

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

Surfactant -- Where Are We in 2003?

Directory of Open Access Journals (Sweden)

JF Lewis

2004-01-01

Full Text Available Surfactant research has progressed over the past several years to the extent that exogenous surfactant administration in patients with the acute respiratory distress syndrome (ARDS is now being evaluated. Unfortunately, clinical responses have been variable, and we now need to take a look at how surfactant is altered in this disease so that more effective treatment strategies can be developed. This review briefly discusses the biophysical and host defense properties of surfactant, the impact of mechanical ventilation (MV on the endogenous surfactant system and the most recent clinical data involving exogenous surfactant administration in patients with ARDS. Discussions regarding future directions of surfactant research both in ARDS and diseases other than acute lung injury are included.

Modulation of the transducer function of Na+,K+-ATPase: new mechanism of heart remodeling.

Science.gov (United States)

Lopatina, Ekaterina V; Kipenko, Anna V; Pasatetskaya, Natalia A; Penniyaynen, Valentina A; Krylov, Boris V

2016-10-01

Endogenous digitalis-like factors were found in the mammalian and human blood. It was the starting point for the elucidation of the new non-pumping function of the Na + ,K + -ATPase. It was previously well known that Na + ,K + -ATPase is a pharmacological target receptor for cardiac glycosides (J.C. Skou. 1957. Biochim. Biophys. Acta, 23: 394-401). We have investigated the trophotropic effects of such agents as ouabain, epinephrine, norepinephrine, atenolol, and comenic acid using the organotypic tissue culture combined with the reconstruction of optical cross sections and confocal microscopy. It was shown that the growth zone of organotypic culture forms a multidimensional structure. Our results indicate that the cardiac glycoside ouabain applied in endogenous concentrations (10 -8 , 10 -10 mol/L) can modulate transducer function of Na + ,K + -ATPase and control the cell growth and proliferation. It was also shown that Src-kinase is involved in "endogenous" ouabain activated intracellular pathways as a series unit. Epinephrine (10 -9 -10 -14 mol/L) and comenic acid (10 -6 -10 -10 mol/L) were demonstrated to modulate the growth of 10- to 12-day-old chicken embryo cardiac tissue explants in a dose-dependent manner. Epinephrine and comenic acid regulate growth and proliferation of the cardiac tissue via receptor-mediated modulation Na + ,K + -ATPase as a signal transducer. The trophotropic effects of the investigated agents specifically control the heart remodeling phenomenon.

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.

Thermodynamics of non-ionic surfactant Triton X-100-cationic surfactants mixtures at the cloud point

International Nuclear Information System (INIS)

Batigoec, Cigdem; Akbas, Halide; Boz, Mesut

2011-01-01

Highlights: → Non-ionic surfactants are used as emulsifier and solubilizate in such as textile, detergent and cosmetic. → Non-ionic surfactants occur phase separation at temperature as named the cloud point in solution. → Dimeric surfactants have attracted increasing attention due to their superior surface activity. → The positive values of ΔG cp 0 indicate that the process proceeds nonspontaneous. - Abstract: This study investigates the effects of gemini and conventional cationic surfactants on the cloud point (CP) of the non-ionic surfactant Triton X-100 (TX-100) in aqueous solutions. Instead of visual observation, a spectrophotometer was used for measurement of the cloud point temperatures. The thermodynamic parameters of these mixtures were calculated at different cationic surfactant concentrations. The gemini surfactants of the alkanediyl-α-ω-bis (alkyldimethylammonium) dibromide type, on the one hand, with different alkyl groups containing m carbon atoms and an ethanediyl spacer, referred to as 'm-2-m' (m = 10, 12, and 16) and, on the other hand, with -C 16 alkyl groups and different spacers containing s carbon atoms, referred to as '16-s-16' (s = 6 and 10) were synthesized, purified and characterized. Additions of the cationic surfactants to the TX-100 solution increased the cloud point temperature of the TX-100 solution. It was accepted that the solubility of non-ionic surfactant containing polyoxyethylene (POE) hydrophilic chain was a maximum at the cloud point so that the thermodynamic parameters were calculated at this temperature. The results showed that the standard Gibbs free energy (ΔG cp 0 ), the enthalpy (ΔH cp 0 ) and the entropy (ΔS cp 0 ) of the clouding phenomenon were found positive in all cases. The standard free energy (ΔG cp 0 ) increased with increasing hydrophobic alkyl chain for both gemini and conventional cationic surfactants; however, it decreased with increasing surfactant concentration.

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.

Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

Directory of Open Access Journals (Sweden)

Yasser A Mahmmoud

Full Text Available Capsazepine (CPZ inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10 of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

Na⁺, K⁺-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

Directory of Open Access Journals (Sweden)

Ali Mobasheri

2012-04-01

Full Text Available Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na⁺, K⁺-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons. Studying the distribution of Na⁺, K⁺-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na⁺, K⁺-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na⁺, K⁺-ATPase in a human chondrocyte cell line (C-20/A4 using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na⁺, K⁺-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na⁺, K⁺-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na⁺, K⁺-ATPase expression and regulation in chondrocytes.

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

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

MICROBIAL SURFACTANTS IN ENVIRONMENTAL TECHNOLOGIES

Directory of Open Access Journals (Sweden)

T. P. Pirog

2015-08-01

Full Text Available It was shown literature and own experimental data concerning the use of microbial surface active glycolipids (rhamno-, sophoro- and trehalose lipids and lipopeptides for water and soil purification from oil and other hydrocarbons, removing toxic heavy metals (Cu2+, Cd2+, Ni2+, Pb2+, degradation of complex pollution (oil and other hydrocarbons with heavy metals, and the role of microbial surfactants in phytoremediation processes. The factors that limit the use of microbial surfactants in environmental technologies are discussed. Thus, at certain concentrations biosurfactant can exhibit antimicrobial properties and inhibit microorganisms destructing xenobiotics. Microbial biodegradability of surfactants may also reduce the effectiveness of bioremediation. Development of effective technologies using microbial surfactants should include the following steps: monitoring of contaminated sites to determine the nature of pollution and analysis of the autochthonous microbiota; determining the mode of surfactant introduction (exogenous addition of stimulation of surfactant synthesis by autochthonous microbiota; establishing an optimal concentration of surfactant to prevent exhibition of antimicrobial properties and rapid biodegradation; research both in laboratory and field conditions.

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.

Dynamic covalent surfactants

NARCIS (Netherlands)

Minkenberg, C.B.

2012-01-01

In this thesis the development of surfactant aggregates with fast exchange dynamics between the aggregated and non-aggregated state is described. Dynamic surfactant exchange plays an important role in natural systems, for instance in cell signaling, cell division, and uptake and release of cargo.

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....

Unaccustomed eccentric contractions impair plasma K+ regulation in the absence of changes in muscle Na+,K+-ATPase content.

Directory of Open Access Journals (Sweden)

Craig A Goodman

Full Text Available The Na+,K+-ATPase (NKA plays a fundamental role in the regulation of skeletal muscle membrane Na+ and K+ gradients, excitability and fatigue during repeated intense contractions. Many studies have investigated the effects of acute concentric exercise on K+ regulation and skeletal muscle NKA, but almost nothing is known about the effects of repeated eccentric contractions. We therefore investigated the effects of unaccustomed maximal eccentric knee extensor contractions on K+ regulation during exercise, peak knee extensor muscle torque, and vastus lateralis muscle NKA content and 3-O-MFPase activity. Torque measurements, muscle biopsies, and venous blood samples were taken before, during and up to 7 days following the contractions in six healthy adults. Eccentric contractions reduced peak isometric muscle torque immediately post-exercise by 26±11% and serum creatine kinase concentration peaked 24 h post-exercise at 339±90 IU/L. During eccentric contractions, plasma [K+] rose during Set 1 and remained elevated at ∼4.9 mM during sets 4-10; this was despite a decline in work output by Set 4, which fell by 18.9% at set 10. The rise in plasma [K+] x work(-1 ratio was elevated over Set 2 from Set 4- Set 10. Eccentric contractions had no effect on muscle NKA content or maximal in-vitro 3-O-MFPase activity immediately post- or up to 7 d post-exercise. The sustained elevation in plasma [K+] despite a decrease in work performed by the knee extensor muscles suggests an impairment in K+ regulation during maximal eccentric contractions, possibly due to increased plasma membrane permeability or to excitation-contraction uncoupling.

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

Treatment with exogenous surfactant stimulates endogenous surfactant synthesis in premature infants with respiratory distress syndrome

NARCIS (Netherlands)

Bunt, JEH; Carnielli, VP; Janssen, DJ; Wattimena, JLD; Hop, WC; Sauer, PJ; Zimmermann, LJI

2000-01-01

Objective: Treatment of preterm infants with respiratory distress syndrome (RDS) with exogenous surfactant has greatly improved clinical outcome. Some infants require multiple doses, and it has not been studied whether these large amounts of exogenous surfactant disturb endogenous surfactant

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

Surfactant nebulisation : lung function, surfactant distribution and pulmonary blood flow distribution in lung lavaged rabbits

NARCIS (Netherlands)

Dijk, Peter H.; Heikamp, A; Bambang Oetomo, Sidarto

1997-01-01

Objective: Surfactant nebulisation is a promising alternative to surfactant instillation in newborns with the respiratory distress syndrome. Although less surfactant is deposited in the lung, it improves gas exchange, probably due to a superior distribution. We hypothesize that a more uniform

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...

Surfactants from petroleum paraffin wax

Energy Technology Data Exchange (ETDEWEB)

Kassem, T.M.; Hussein, M.H.; El Sayed, A.S.

Paraffin wax from Egyptian petroleum was purified and then oxidized to fatty acids which were esterified to form their methyl esters, fractionated and then hydrolysed. The obtained fatty acids were converted into the corresponding primary amines which were converted with ethylene oxide to form nonionic surfactants. The prepared primary amines were also converted into tertiary amines and then converted into cationic surfactants through condensation with benzyl chloride or 1-chloromethylnaphthalene. Also, amine oxide surfactants were prepared by oxidation of the tertiary amines with hydrogen peroxide. The surface active properties of all the prepared surfactants were determined, and the effect of their chemical structure on the surfactant properties are discussed in this paper.

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.

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.

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

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

Acute Pathophysiological Effects of Intratracheal Instillation of Budesonide and Exogenous Surfactant in a Neonatal Surfactant-depleted Piglet Model

Directory of Open Access Journals (Sweden)

Chia-Feng Yang

2010-08-01

Conclusions: Intratracheal instillation of surfactant or surfactant plus budesonide can improve oxygenation and pulmonary histologic outcome in neonatal surfactant-depleted lungs. The additional use of budesonide does not disturb the function of the exogenous surfactant. Intratracheal administration of a corticosteroid combined with surfactant may be an effective method for alleviating local pulmonary inflammation in severe RDS.

Surfactant flooding of diesel-contaminated soils

International Nuclear Information System (INIS)

Peters, R.W.; Montemagno, C.D.; Shem, L.; Lewis, B.A.

1991-01-01

At one installation, approximately 60,000 gallons of No. 2 diesel fuel leaked into the subsurface environment, with contamination at depths of 6 to 34 m below the surface. Argonne National Laboratory was contracted to perform treatability studies for site remediation. The treatability studies focused on four separate phases: (1) leachability studies on the various contaminated soil borings, (2) air stripping studies, (3) bioremediation studies, and (4) surfactant screening/surfactant flooding studies. This paper summarizes the fourth phase of this research program after initial surfactant screening of 21 surfactants. Three of the surfactants were used for the surfactant flooding studies; the results from that phase of the research program are described

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

A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes.

Directory of Open Access Journals (Sweden)

Antonia E G Booth

Full Text Available The mechanism(s by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1 as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP, is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.

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...

Estrogen, progesterone, and genistein differentially regulate levels of expression of α-, β-, and γ-epithelial sodium channel (ENaC) and α-sodium potassium pump (Na⁺/K⁺-ATPase) in the uteri of sex steroid-deficient rats.

Science.gov (United States)

Chinigarzadeh, Asma; Muniandy, Sekaran; Salleh, Naguib

2015-10-01

Estrogen, progesterone, and genistein could induce changes in uterine fluid volume and Na(+) concentration. Progesterone upregulates expression of epithelial sodium channel (ENaC) and Na(+)/K(+)-ATPase which contributed toward these changes. However, effects of estrogen and genistein were unknown. This study therefore investigated changes in expression of these proteins in the uterus under estrogen, progesterone, and genistein influences to further understand mechanisms underlying sex steroids and phytoestrogen effects on uterine fluid Na(+) regulation. In this study, uteri of ovariectomized female rats receiving 7-day treatment with genistein (25, 50, and 100 mg/kg/day), estrogen (0.8 × 10(-4) mg/kg/day), or progesterone (4 mg/kg/day) were harvested, and expression levels of α-, β-, and γ-ENaC proteins and messenger RNAs (mRNAs) and α-Na(+)/K(+)-ATPase protein were determined by Western blotting (proteins) and real-time polymerase chain reaction (mRNA). Meanwhile, distribution of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase proteins in the uterus was identified by immunohistochemistry. Our findings indicated that expression of α-, β-, and γ-ENaC proteins and mRNAs and α-Na(+)/K(+)-ATPase protein were enhanced under progesterone influence. Lower expressions were noted under estrogen and genistein influences compared to progesterone. Under estrogen, progesterone, and genistein influences, α- and β-ENaC were distributed at apical membrane and γ-ENaC was distributed at apical and basolateral membranes of uterine luminal epithelia. Under progesterone influence, α-Na(+)/K(+)-ATPase was highly expressed at basolateral membrane. In conclusion, high expression of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase under progesterone influence would contribute toward increased uterine fluid Na(+) reabsorption, whereas lesser expression of these proteins under estrogen and genistein influences would contribute toward lower reabsorption of uterine fluid Na

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)

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.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

Science.gov (United States)

Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

2014-09-15

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. Copyright © 2014 the American Physiological Society.

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

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.

7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

Science.gov (United States)

Duran, M J; Pierre, S V; Lesnik, P; Pieroni, G; Bourdeaux, M; Dignat-Georges, F; Sampol, J; Maixent, J M

2010-11-09

As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

Developmental regulation of chicken surfactant protein A and its localization in lung

DEFF Research Database (Denmark)

Zhang, Weidong; Cuperus, Tryntsje; van Dijk, Albert

2016-01-01

Surfactant Protein A (SP-A) is a collagenous C-type lectin (collectin) that plays an important role in the early stage of the host immune response. In chicken, SP-A (cSP-A) is expressed as a 26 kDa glycosylated protein in the lung. Using immunohistochemistry, cSP-A protein was detected mainly in ...

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).

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

Fluorescent visualization of a spreading surfactant

Energy Technology Data Exchange (ETDEWEB)

Fallest, David W; Lichtenberger, Adele M; Fox, Christopher J; Daniels, Karen E, E-mail: kdaniel@ncsu.ed [Department of Physics, North Carolina State University, Raleigh, NC 27695 (United States)

2010-07-15

The spreading of surfactants on thin films is an industrially and medically important phenomenon, but the dynamics are highly nonlinear and visualization of the surfactant dynamics has been a long-standing experimental challenge. We perform the first quantitative, spatiotemporally resolved measurements of the spreading of an insoluble surfactant on a thin fluid layer. During the spreading process, we directly observe both the radial height profile of the spreading droplet and the spatial distribution of the fluorescently tagged surfactant. We find that the leading edge of a spreading circular layer of surfactant forms a Marangoni ridge in the underlying fluid, with a trough trailing the ridge as expected. However, several novel features are observed using the fluorescence technique, including a peak in the surfactant concentration that trails the leading edge, and a flat, monolayer-scale spreading film that differs from concentration profiles predicted by current models. Both the Marangoni ridge and the surfactant leading edge can be described to spread as R{approx}t{sup {delta}}. We find spreading exponents {delta}{sub H}{approx}0.30 and {delta}{sub {Gamma}}{approx}0.22 for the ridge peak and surfactant leading edge, respectively, which are in good agreement with theoretical predictions of {delta}=1/4. In addition, we observe that the surfactant leading edge initially leads the peak of the Marangoni ridge, with the peak later catching up to the leading edge.

Rhamnolipids--next generation surfactants?

Science.gov (United States)

Müller, Markus Michael; Kügler, Johannes H; Henkel, Marius; Gerlitzki, Melanie; Hörmann, Barbara; Pöhnlein, Martin; Syldatk, Christoph; Hausmann, Rudolf

2012-12-31

The demand for bio-based processes and materials in the petrochemical industry has significantly increased during the last decade because of the expected running out of petroleum. This trend can be ascribed to three main causes: (1) the increased use of renewable resources for chemical synthesis of already established product classes, (2) the replacement of chemical synthesis of already established product classes by new biotechnological processes based on renewable resources, and (3) the biotechnological production of new molecules with new features or better performances than already established comparable chemically synthesized products. All three approaches are currently being pursued for surfactant production. Biosurfactants are a very promising and interesting substance class because they are based on renewable resources, sustainable, and biologically degradable. Alkyl polyglycosides are chemically synthesized biosurfactants established on the surfactant market. The first microbiological biosurfactants on the market were sophorolipids. Of all currently known biosurfactants, rhamnolipids have the highest potential for becoming the next generation of biosurfactants introduced on the market. Although the metabolic pathways and genetic regulation of biosynthesis are known qualitatively, the quantitative understanding relevant for bioreactor cultivation is still missing. Additionally, high product titers have been exclusively described with vegetable oil as sole carbon source in combination with Pseudomonas aeruginosa strains. Competitive productivity is still out of reach for heterologous hosts or non-pathogenic natural producer strains. Thus, on the one hand there is a need to gain a deeper understanding of the regulation of rhamnolipid production on process and cellular level during bioreactor cultivations. On the other hand, there is a need for metabolizable renewable substrates, which do not compete with food and feed. A sustainable bioeconomy approach should

Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells

DEFF Research Database (Denmark)

Friis, U G; Praetorius, Birger Hans; Knudsen, T

1997-01-01

1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use...

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.

Surfactant screening of diesel-contaminated soil

International Nuclear Information System (INIS)

Peters, R.W.; Montemagno, C.D.; Shem, L.; Lewis, B.-A.

1992-01-01

At one installation in California, approximately 60,000 gal of No. 2 diesel fuel leaked into the subsurface environment, resulting in contamination at depths from 6 to 34 m below the surface. Argonne National Laboratory was contracted to perform treatability studies for site remediation. This paper summarizes a surfactant screening/surfactant flooding research program in which 22 surfactants were screened for their effectiveness in mobilizing the organics from the contaminated soil prior to bioremediation. Anionic surfactants resulted in the greatest degree of diesel mobilization. The most promising surfactants will be employed on contaminated soil samples obtained from the site

Quantitative determination of Na+-K+-ATPase and other sarcolemmal components in muscle cells

International Nuclear Information System (INIS)

Hansen, O.; Clausen, T.

1988-01-01

A recurring problem in the characterization of plasma membrane enzymes in tissues and cells is whether the samples tested are representative for the entire population of enzyme molecules present in the starting material. Measurements of [ 3 H]-ouabain binding, enzyme activity, and maximum transport capacity all indicate that the concentration of Na + -K + pumps in mammalian skeletal muscle is high. Studies on Na + -K + -ATPase activity in isolated sarcolemma, however, generally give little or no information on total cellular enzyme concentration. Due to the low and variable enzyme recovery, such subcellular preparations may, therefore, give misleading data on factors regulating Na + -K + -ATPase in heart and skeletal muscle cells. As the same isolation and purification procedures are used for the study of other sarcolemmal components, this inadequate recovery has general implications for statements on regulatory changes in the sarcolemmal composition of muscle cells. On the other hand, complete quantification of Na + -K + -ATPase in muscle tissue can now be achieved using simple procedures and the entire material. Recent studies have shown that regulatory changes in the entire population of Na + -K + pumps in muscle can be quantified in measurements of [ 3 H]-ouabain binding, K + -activated 3-O-methylfluorescein phosphatase activity, as well as maximum ouabain suppressible Na + -K + transport capacity

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...

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

Surfactant nebulization versus instillation during high frequency ventilation in surfactant-deficient rabbits

NARCIS (Netherlands)

Dijk, Peter H.; Heikamp, A; Bambang Oetomo, Sidarto

Surfactant nebulization improves lung function at low alveolar doses of surfactant. However, efficiency of nebulization is low, and lung deposition seems to depend on lung aeration. High frequency ventilation (HFV) has been shown to improve lung aeration. We hypothesize that the combination of HFV

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.

BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination.

Science.gov (United States)

Shim, Do-Wan; Shin, Woo-Young; Yu, Sang-Hyeun; Kim, Byung-Hak; Ye, Sang-Kyu; Koppula, Sushruta; Won, Hyung-Sik; Kang, Tae-Bong; Lee, Kwang-Ho

2017-11-08

The ATPase activity of NLRP3 has pivotal role in inflammasome activation and is recognized as a good target for the development of the NLRP3 inflammasome-specific inhibitor. However, signals in the vicinity of the ATPase activity of NLRP3 have not been fully elucidated. Here, we demonstrate NLRP3 inflammasome-specific action of a benzoxathiole derivative, BOT-4-one. BOT-4-one exhibited an inhibition of NLRP3 inflammasome activation, which was attributable to its alkylating capability to NLRP3. In particular, the NLRP3 alkylation by BOT-4-one led to an impaired ATPase activity of NLRP3, thereby obstructing the assembly of the NLRP3 inflammasome. Additionally, we found that NLRP3 alkylators, including BOT-4-one, enhance the ubiquitination level of NLRP3, which might also contribute to the inhibition of NLRP3 inflammasome activation. Finally, BOT-4-one appeared to be superior to other known NLRP3 alkylators in inhibiting the functionality of the NLRP3 inflammasome and its resulting anti-inflammatory activity was confirmed in vivo using a monosodium urate-induced peritonitis mouse model. Collectively, the results suggest that NLRP3 alkylators function by inhibiting ATPase activity and increasing the ubiquitination level of NLRP3, and BOT-4-one could be the type of NLRP3 inhibitor that may be potentially useful for the novel development of a therapeutic agent in controlling NLRP3 inflammasome-related diseases.

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

Science.gov (United States)

Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

2015-10-01

Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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...

Solubilization of Hydrophobic Dyes in Surfactant Solutions

Directory of Open Access Journals (Sweden)

Ali Reza Tehrani-Bagha

2013-02-01

Full Text Available In this paper, the use of surfactants for solubilization of hydrophobic organic dyes (mainly solvent and disperse dyes has been reviewed. The effect of parameters such as the chemical structures of the surfactant and the dye, addition of salt and of polyelectrolytes, pH, and temperature on dye solubilization has been discussed. Surfactant self-assemble into micelles in aqueous solution and below the concentration where this occurs—the critical micelle concentration (CMC—there is no solubilization. Above the CMC, the amount of solubilized dye increases linearly with the increase in surfactant concentration. It is demonstrated that different surfactants work best for different dyes. In general, nonionic surfactants have higher solubilization power than anionic and cationic surfactants. It is likely that the reason for the good performance of nonionic surfactants is that they allow dyes to be accommodated not only in the inner, hydrocarbon part of the micelle but also in the headgroup shell. It is demonstrated that the location of a dye in a surfactant micelle can be assessed from the absorption spectrum of the dye-containing micellar solution.

Evaluation of the presence of major anionic surfactants in marine sediments.

Science.gov (United States)

Cantarero, S; Camino-Sánchez, F J; Zafra-Gómez, A; Ballesteros, O; Navalón, A; Vílchez, J L; Verge, C; Reis, M S; Saraiva, P M

2012-03-01

The contamination of aquatic environments has become the focus of increasing regulation and public concern due to their potential and unknown negative effects on the ecosystems. The present work develops a monitoring and statistical study, based on the analysis of variance test (ANOVA) and the multivariable analysis, both for insoluble soap and LAS in order to compare the behavior of different anionic surfactants in this environmental compartment. First, a novel and successfully validated methodology to analyze insoluble soap in these samples is developed. The matrix effect and the comparison of different extraction techniques were also performed. The optimized analytical methodologies were applied to 48 representative samples collected from the Almeria Coast (Spain) and then a statistical analysis to correlate anionic surfactant concentration and several variables associated with marine sediment samples was also developed. The results obtained showed relevant conclusions related to the environmental behavior of anionic surfactants in marine sediments. Copyright © 2011 Elsevier Ltd. All rights reserved.

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...

NMR study of the dynamics of cationic gemini surfactant 14-2-14 in mixed solutions with conventional surfactants.

Science.gov (United States)

Jiang, Yan; Lu, Xing-Yu; Chen, Hong; Mao, Shi-Zhen; Liu, Mai-Li; Luo, Ping-Ya; Du, You-Ru

2009-06-18

Three kinds of conventional surfactants, namely, two nonionic surfactants [polyethylene glycol (23) lauryl ether (Brij-35) and Triton X-100 (TX-100)], one cationic surfactant [n-tetradecyltrimethyl ammonium bromide (TTAB)], and an anionic surfactant [sodium n-dodecyl sulfate (SDS)}, were mixed into the quaternary ammonium gemini surfactant [C(14)H(29)N(+)(CH(3))(2)](2)(CH(2))(2).2Br(-) (14-2-14) in aqueous solution. The exchange rate constants between 14-2-14 molecules in the mixed micelles and those in the bulk solution were detected using two nuclear magnetic resonance (NMR) methods: one-dimensional (1D) line shape analysis and two-dimensional (2D) exchange spectroscopy (EXSY). The results obtained from these two methods were consistent. Both showed that mixing a nonionic conventional surfactant, either Brij-35 or TX-100, enhanced the exchange process between the 14-2-14 molecules in the mixed micelles and those in the bulk solution. In contrast, the anionic surfactant SDS and the cationic surfactant TTAB slowed the process slightly.

Beneficial renal and pancreatic phenotypes in a mouse deficient in FXYD2 regulatory subunit of Na,K-ATPase

Directory of Open Access Journals (Sweden)

Elena eArystarkhova

2016-03-01

Full Text Available The fundamental role of Na,K-ATPase in eukaryotic cells calls for complex and efficient regulation of its activity. Besides alterations in gene expression and trafficking, kinetic properties of the pump are modulated by reversible association with single span membrane proteins, the FXYDs. Seven members of the family are expressed in a tissue-specific manner, affecting pump kinetics in all possible permutations. This mini-review focuses on functional properties of FXYD2 studied in transfected cells, and on noteworthy and unexpected phenotypes discovered in a Fxyd2-/- mouse. FXYD2, the gamma subunit, reduces activity of Na,K-ATPase either by decreasing affinity for Na+, or reducing Vmax. FXYD2 mRNA splicing and editing provide another layer for regulation of Na,K-ATPase. In kidney of knockouts, there was elevated activity for Na,K-ATPase and for NCC and NKCC2 apical sodium transporters. That should lead to sodium retention and hypertension, however, the mice were in sodium balance and normotensive. Adult Fxyd2-/- mice also exhibited a mild pancreatic phenotype with enhanced glucose tolerance, elevation of circulating insulin, but no insulin resistance. There was an increase in beta cell proliferation and beta cell mass that correlated with activation of the PI3K-Akt pathway. The Fxyd2-/- mice are thus in a highly desirable state: the animals are resistant to Na+ retention, and showed improved glucose control, i.e. they display favorable metabolic adaptations to protect against development of salt-sensitive hypertension and diabetes. Investigation of the mechanisms of these adaptations in the mouse has the potential to unveil a novel therapeutic FXYD2-dependent strategy.

The beta1 subunit of the Na,K-ATPase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 (MLC1) in brain astrocytes: new insights into MLC pathogenesis.

Science.gov (United States)

Brignone, Maria S; Lanciotti, Angela; Macioce, Pompeo; Macchia, Gianfranco; Gaetani, Matteo; Aloisi, Francesca; Petrucci, Tamara C; Ambrosini, Elena

2011-01-01

Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the β1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-β1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.

Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R. (Brigham and Women' s Hospital, Boston, MA (USA))

1989-12-01

Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake.

Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

International Nuclear Information System (INIS)

Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R.

1989-01-01

Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake

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.

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.

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.)

Identification of a Nicotiana plumbaginifolia plasma membrane H(+)-ATPase gene expressed in the pollen tube.

Science.gov (United States)

Lefebvre, Benoit; Arango, Miguel; Oufattole, Mohammed; Crouzet, Jérôme; Purnelle, Bénédicte; Boutry, Marc

2005-08-01

In Nicotiana plumbaginifolia, plasma membrane H(+)-ATPases (PMAs) are encoded by a gene family of nine members. Here, we report on the characterization of a new isogene, NpPMA5 (belonging to subfamily IV), and the determination of its expression pattern using the beta-glucuronidase (gusA) reporter gene. pNpPMA5-gusA was expressed in cotyledons, in vascular tissues of the stem (mainly in nodal zones), and in the flower and fruit. In the flower, high expression was found in the pollen tube after in vitro or in vivo germination. Northern blotting analysis confirmed that NpPMA5 was expressed in the pollen tube contrary to NpPMA2 (subfamily I) or NpPMA4 (subfamily II), two genes highly expressed in other tissues. The subcellular localization of PM H(+)-ATPase in the pollen tube was analyzed by immunocytodecoration. As expected, this enzyme was localized to the plasma membrane. However, neither the tip nor the base of the pollen tube was labeled, showing an asymmetrical distribution of this enzyme. This observation supports the hypothesis that the PM H(+)-ATPase is involved in creating the pH gradient that is observed along the pollen tube and is implicated in cell elongation. Compared to other plant PM H(+)-ATPases, the C-terminal region of NpPMA5 is shorter by 26 amino acid residues and is modified in the last 6 residues, due to a sequence rearrangement, which was also found in the orthologous gene of Nicotiana glutinosa, a Nicotiana species distant from N. plumbaginifolia and Petunia hybrida and Lycopersicon esculentum, other Solanacae species. This modification alters part of the PM H(+)-ATPase regulatory domain and raises the question whether this isoform is still regulated.

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

Science.gov (United States)

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.

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

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.

Pulmonary clearance of {sup 99m}Tc-DTPA in experimental surfactant dysfunction treated with surfactant installation

Energy Technology Data Exchange (ETDEWEB)

Nilsson, K.; John, J.; Lachmann, B.; Robertson, B.; Wollmer, P.

1997-02-01

Background: Breakdown of the alveolo-capillary barrier is a characteristic feature of respiratory distress syndrome. Restoration of alveolo-capillary barrier function may be an important aspect of surfactant replacement therapy. We examined the effect of surfactant installation on alveolo-capillary barrier function in an experimental model of surfactant dysfunction by measuring pulmonary clearance of {sup 99m}Tc-DTPA. Methods: Nineteen rabbits were tracheotomized and mechanically ventilated. Surfactant dysfunction was induced by administration of a synthetic detergent in aerosol form. Detergent was given to 13 rabbits; seven rabbits were then treated with installation of natural surfactant, whereas six rabbits received saline. Six rabbits were used as untreated controls. An aerosol of {sup 99m}Tc-DTPA was administered to all animals and the pulmonary clearance was measured with a gamma camera. Results: {sup 99m}Tc-DTPA cleared from the lungs with a half-life of 71{+-}22 min in the control animals, 21.4{+-}7.4 min in the surfactant-treated animals and 5.8{+-}1.5 min in the saline-treated animals. The difference in half-life between groups was highly significant (P<0.001). There was no change in arterial oxygenation or compliance in controls or in animals treated with saline. In animals treated with surfactant, a small transient reduction in arterial oxygen tension and a more long-standing reduction in compliance were observed. Conclusion: Surfactant treatment thus significantly attenuated the effect of detergent treatment but did not restore alveolo-capillary transfer of {sup 99m}Tc-DTPA to normal. (AU) 26 refs.

Surfactant protein A and surfactant protein D variation in pulmonary disease

DEFF Research Database (Denmark)

Sørensen, Grith Lykke; Husby, Steffen; Holmskov, Uffe

2007-01-01

Surfactant proteins A (SP-A) and D (SP-D) have been implicated in pulmonary innate immunity. The proteins are host defense lectins, belonging to the collectin family which also includes mannan-binding lectin (MBL). SP-A and SP-D are pattern-recognition molecules with the lectin domains binding...... lavage and blood have indicated associations with a multitude of pulmonary inflammatory diseases. In addition, accumulating evidence in mouse models of infection and inflammation indicates that recombinant forms of the surfactant proteins are biologically active in vivo and may have therapeutic potential...... in controlling pulmonary inflammatory disease. The presence of the surfactant collectins, especially SP-D, in non-pulmonary tissues, such as the gastrointestinal tract and genital organs, suggest additional actions located to other mucosal surfaces. The aim of this review is to summarize studies on genetic...

Activation of the Na+,K+-ATPase in Narcine brasiliensis

International Nuclear Information System (INIS)

Blum, H.; Nioka, Shoko; Johnson, R.G. Jr.

1990-01-01

The in vivo activation and turnover rates of the sodium pump (Na + ,K + -ATPase) were investigated in the electrocytes of the electric organ of the elasmobranch Narcine brasiliensis. The Narcine electric organ appears to be an excellent model for the study of sodium pump activation in an excitable tissue. The sodium transmembrane gradient and high-energy phosphagens were concurrently measured by 23 Na and 31 P NMR spectroscopy. The resting electric organ, which depends primarily on anaerobic metabolism displays a high concentration of phosphocreatin (PCr). It has an intracellular sodium concentration ([Na + ] i ) of 20±10 milliequivalents/liter as estimated by NMR. Electrical stimulation of the nerves innervating the electric organ results in an increase in [Na + ] i in the electrolyte and rapid depletion of PCr. Ouabain causes an 85% decrease in utilization of high-energy phosphagens, indicating that rapid PCr turnover in this tissue is mainly due to Na + ,K + -ATPase activity. From these data the authors can determine that the rate of sodium pump turnover increases by >3 orders of magnitude within several hundred milliseconds. The authors conclude that cholinergic stimulation of the electric organ causes a rapid and extremely large increase in sodium pump turnover, which is regulated predominantly by factors other than [Na + ] i

Adsorption of dimeric surfactants in lamellar silicates

Energy Technology Data Exchange (ETDEWEB)

Balcerzak, Mateusz; Pietralik, Zuzanna [Department of Macromolecular Physics, Faculty of Physics, A. Mickiewicz University, Umultowska 85, 61-614 Poznań (Poland); Domka, Ludwik [Department of Metalorganic Chemistry, Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznań (Poland); Skrzypczak, Andrzej [Institute of Chemical Technology, Poznań University of Technology, Berdychowo 4, 60-965 Poznań (Poland); Kozak, Maciej, E-mail: mkozak@amu.edu.pl [Department of Macromolecular Physics, Faculty of Physics, A. Mickiewicz University, Umultowska 85, 61-614 Poznań (Poland)

2015-12-01

Highlights: • The intercalation of dimeric surfactants changed the morphology of MMT samples. • XRD indicated structures formed by surfactant molecules in interlayer space. • The four-step thermal decomposition of dimeric surfactant, confirms intercalation. - Abstract: The adsorption of different types of cationic surfactants in lamellar silicates changes their surface character from hydrophilic to hydrophobic. This study was undertaken to obtain lamellar silicates modified by a series of novel dimeric (gemini) surfactants of different length alkyl chains and to characterise these organophilised materials. Synthetic sodium montmorillonite SOMASIF® ME 100 (M) and enriched bentonite of natural origin (Nanoclay – hydrophilic bentonite®) were organophilised with dimeric (gemini) surfactants (1,1′-(1,4-butanediyl)bis(alkoxymethyl)imidazolium dichlorides). As a result of surfactant molecule adsorption in interlamellar space, the d-spacing (d{sub 001}) increased from 0.97 nm (for the anhydrous structure) to 2.04 nm. A Fourier transform infrared spectroscopy (FTIR) analysis of the modified systems reveals bands assigned to the stretching vibrations of the CH{sub 2} and CH{sub 3} groups and the scissoring vibrations of the NH group from the structure of the dimeric surfactants. Thermogravimetric (TG) and derivative thermogravimetric (DTG) studies imply a four-stage process of surfactant decomposition. Scanning electron microscopy (SEM) images provide information on the influence of dimeric surfactant intercalation into the silicate structures. Particles of the modified systems show a tendency toward the formation of irregularly shaped agglomerates.

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.

Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

Directory of Open Access Journals (Sweden)

Brian Panicucci

2017-04-01

Full Text Available The mitochondrial (mt FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF, but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF, which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm. Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1 binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1, but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

Biodegradation of surfactant bearing wastes

International Nuclear Information System (INIS)

Chitra, S.; Chandran, S.; Sasidhar, P.; Lal, K.B.; Amalraj, R.V.

1991-01-01

In nuclear industry, during decontamination of protective wears and contaminated materials, detergents are employed to bring down the level of radioactive contamination within safe limits. However, the surfactant present in these wastes interferes in the chemical treatment process, reducing the decontamination factor. Biodegradation is an efficient and ecologically safe method for surfactant removal. A surfactant degrading culture was isolated and inoculated separately into simulated effluents containing 1% yeast extract and 5-100 ppm sodium lauryl sulphate (SLS) and 1% yeast extract and 5-100 ppm of commercial detergent respectively. The growth of the bacterial culture and the degradation characteristics of the surfactant in the above effluents were monitored under both dynamic and static conditions. (author). 6 refs., 6 figs., 1 tab

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

Science.gov (United States)

Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

2018-01-31

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

Estimation hydrophilic-lipophilic balance number of surfactants

Energy Technology Data Exchange (ETDEWEB)

Pawignya, Harsa, E-mail: harsa-paw@yahoo.co.id [Chemical Engineering Department Diponegoro University (Indonesia); Chemical Engineering Departement University of Pembangunan Nasional Yogyakarta (Indonesia); Prasetyaningrum, Aji, E-mail: ajiprasetyaningrum@gmail.com; Kusworo, Tutuk D.; Pramudono, Bambang, E-mail: Pramudono2004@yahoo.com [Chemical Engineering Department Diponegoro University (Indonesia); Dyartanti, Endah R. [Chemical Engineering Department Diponegoro University (Indonesia); Chemical Enginering Departement Sebelas Maret University (Indonesia)

2016-02-08

Any type of surfactant has a hydrophilic-lipophilic balance number (HLB number) of different. There are several methods for determining the HLB number, with ohysical properties of surfactant (solubility cloud point and interfacial tension), CMC methods and by thermodynamics properties (Free energy Gibbs). This paper proposes to determined HLB numbers from interfelation methods. The result of study indicated that the CMC method described by Hair and Moulik espesially for nonionic surfactant. The application of exess Gibbs free energy and by implication activity coefficient provides the ability to predict the behavior of surfactants in multi component mixtures of different concentration. Determination of HLB number by solubility and cloud point parameter is spesific for anionic and nonionic surfactant but this methods not available for cationic surfactants.

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.

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

International Nuclear Information System (INIS)

Mathur, Chhavi; Savithri, Handanahal S.

2012-01-01

Highlights: ► Pepper vein banding potyvirus VPg harbors Walker motifs. ► VPg exhibits ATPase activity in the presence of NIa-Pro. ► Plausible structural and functional interplay between VPg and NIa-Pro. ► Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

Degradation of surfactants by sono-irradiation

International Nuclear Information System (INIS)

Ashokkumar, M.; Grieser, F.; Vinodgopal, K.

2000-01-01

Full text: The ultrasound induced decomposition of a commercially available polydisperse nonylphenol ethoxylate surfactant (Teric GN9) has been investigated. Nearly 90% mineralization and/or degradation into volatile products of the surfactant is achieved after sonication for 24 hours. Ultrasound has been found to be a useful tool to achieve a number of chemical processes. Linear and branched alkyl benzene sulfonates and alkyl nonylphenol ethoxylates are widely used surfactants which accumulated in the environment and contribute to a well-recognised pollution problem. We have investigated the use of ultrasound in the degradation of both types of surfactants with the aim of understanding the mechanism of degradation in order to optimise the decomposition process. In this presentation, we report on the sonochemical degradation of Teric GN9- polydisperse, a nonylphenol ethoxylate with an average of 9 ethylene oxide units. The ultrasound unit used for the degradation studies of the surfactant solutions was an Allied Signal (ELAC Nautik) RF generator and transducer with a plate diameter of 54.5 mm operated at 363 kHz in continuous wave mode at an intensity of 2 W/cm 2 . Ultrasound induced cavitation events generate primary radicals inside gas/vapour filled bubbles. Due to the extreme conditions (T ∼ 5000 K; P ∼ 100 atm) generated within the collapsing bubble, H and OH radicals are produced by the homolysis of water molecules, if water is the medium of sonication. These primary radicals attack the surfactant molecules adsorbed at the bubble/water interface. The initial rate of reaction of the surfactant was found to be dependent on the monomer concentration in solution below and above the critical micelle concentration of the surfactants. This result strongly suggests that the initial radical attack on the surfactants occurs at the cavitation bubble/solution interface, followed by oxidative decomposition and pyrolysis of volatile fragments of the surfactant within

Poly(ethylene oxide) surfactant polymers.

Science.gov (United States)

Vacheethasanee, Katanchalee; Wang, Shuwu; Qiu, Yongxing; Marchant, Roger E

2004-01-01

We report on a series of structurally well-defined surfactant polymers that undergo surface-induced self-assembly on hydrophobic biomaterial surfaces. The surfactant polymers consist of a poly(vinyl amine) backbone with poly(ethylene oxide) and hexanal pendant groups. The poly(vinyl amine) (PVAm) was synthesized by hydrolysis of poly(N-vinyl formamide) following free radical polymerization of N-vinyl formamide. Hexanal and aldehyde-terminated poly(ethylene oxide) (PEO) were simultaneously attached to PVAm via reductive amination. Surfactant polymers with different PEO:hexanal ratios and hydrophilic/hydrophobic balances were prepared, and characterized by FT-IR, 1H-NMR and XPS spectroscopies. Surface active properties at the air/water interface were determined by surface tension measurements. Surface activity at a solid surface/water interface was demonstrated by atomic force microscopy, showing epitaxially molecular alignment for surfactant polymers adsorbed on highly oriented pyrolytic graphite. The surfactant polymers described in this report can be adapted for simple non-covalent surface modification of biomaterials and hydrophobic surfaces to provide highly hydrated interfaces.

Surfactant screening of diesel-contaminated soil

International Nuclear Information System (INIS)

Peters, R.W.; Shem, L.; Montemagno, C.D.; Lewis, B.

1991-01-01

At one installation, approximately 60,000 gal of No. 2 diesel fuel leaked into the subsurface environment, with contamination at depths of 6 to 34 m below the surface. Argonne National Laboratory was contracted to perform treatability studies for site remediation. The treatability studies focused on four separate phases: (1) leachability studies on the various contaminated soil borings, (2) air stripping studies, (3) bioremediation studies, and (4) surfactant screening/surfactant flooding studies. This paper summarizes the fourth phase of the research program in which 21 surfactants were screened for possible use to mobilize the organics from the contaminated soil prior to bioremediation. Anionic surfactants resulted in the greatest degree of diesel mobilization. The most promising surfactants will be employed on actual contaminated soil samples obtained from the site

Alveolar Thin Layer Flows and Surfactant Dynamics

Science.gov (United States)

Roumie, Ahmad; Jbaily, Abdulrahman; Szeri, Andrew J.

2017-11-01

Pulmonary surfactants play a vital role in everyday respiration. They regulate surface tension in the lungs by diffusing through the hypophase, a liquid layer that lines the interior surface of the alveoli, and adsorbing to the existing air-fluid interface. This decreases the equilibrium surface tension value by as much as a factor of 3, minimizing breathing effort and preventing lung collapse at the end of exhalation. Given that the hypophase thickness h lies within the range 0.1 μm < h <0.5 μm , and that the average alveolar radius R is 100 μm , for some purposes the hypophase may usefully be modeled as a fluid layer on a flat sheet representing the alveolar wall. Moreover, because of the large aspect ratio, the lubrication approximation can be applied. The aim of the present work is to study the interaction between the straining of the alveolar wall and the fluid flow in the hypophase. The analysis is governed by the relative magnitudes of the time scales of surfactant diffusion, adsorption, desorption, viscous dissipation and sheet straining. Cases of particular interest include non-uniform surfactant concentration at the interface, leading to Marangoni flows and a non-uniform hypophase thickness profile. The analytical formulation and numerical simulations are presented. This work is motivated by a need to understand alveolar deformation during breathing, and to do so in a way that derives from improved understanding of the fluid mechanics of the problem.

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.

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

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...

Dilute Surfactant Methods for Carbonate Formations

Energy Technology Data Exchange (ETDEWEB)

Kishore K. Mohanty

2006-02-01

There are many fractured carbonate reservoirs in US (and the world) with light oil. Waterflooding is effective in fractured reservoirs, if the formation is water-wet. Many fractured carbonate reservoirs, however, are mixed-wet and recoveries with conventional methods are low (less than 10%). The process of using dilute anionic surfactants in alkaline solutions has been investigated in this work for oil recovery from fractured oil-wet carbonate reservoirs both experimentally and numerically. This process is a surfactant-aided gravity drainage where surfactant diffuses into the matrix, lowers IFT and contact angle, which decrease capillary pressure and increase oil relative permeability enabling gravity to drain the oil up. Anionic surfactants have been identified which at dilute concentration of 0.05 wt% and optimal salinity can lower the interfacial tension and change the wettability of the calcite surface to intermediate/water-wet condition as well or better than the cationic surfactant DTAB with a West Texas crude oil. The force of adhesion in AFM of oil-wet regions changes after anionic surfactant treatment to values similar to those of water-wet regions. The AFM topography images showed that the oil-wetting material was removed from the surface by the anionic surfactant treatment. Adsorption studies indicate that the extent of adsorption for anionic surfactants on calcite minerals decreases with increase in pH and with decrease in salinity. Surfactant adsorption can be minimized in the presence of Na{sub 2}CO{sub 3}. Laboratory-scale surfactant brine imbibition experiments give high oil recovery (20-42% OOIP in 50 days; up to 60% in 200 days) for initially oil-wet cores through wettability alteration and IFT reduction. Small (<10%) initial gas saturation does not affect significantly the rate of oil recovery in the imbibition process, but larger gas saturation decreases the oil recovery rate. As the core permeability decreases, the rate of oil recovery reduces

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.

Surfactant gene polymorphisms and interstitial lung diseases

Directory of Open Access Journals (Sweden)

Pantelidis Panagiotis

2001-11-01

Full Text Available Abstract Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs. Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.

[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.

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.

Formation and characterization of zein-propylene glycol alginate-surfactant ternary complexes: Effect of surfactant type.

Science.gov (United States)

Dai, Lei; Sun, Cuixia; Wei, Yang; Zhan, Xinyu; Mao, Like; Gao, Yanxiang

2018-08-30

In this study, zein, propylene glycol alginate (PGA) and surfactant ternary complexes were fabricated by antisolvent co-precipitation method. Two types of surfactants (rhamnolipid and lecithin) were applied to generate zein-PGA-rhamnolipid (Z-P-R) and zein-PGA-lecithin (Z-P-L) ternary complexes, respectively. Results showed that the surfactant types significantly affected the properties of ternary complexes. The formation of ternary complexes was mainly due to the non-covalent interactions such as hydrogen bonding, electrostatic interaction and hydrophobic interactions among zein, PGA and surfactants. Moreover, the thermal stability of ternary complexes was enhanced with increasing the levels of both surfactants. Notably, ternary complex dispersions exhibited better stability against pH from 2 to 8. Furthermore, a compact network structure was observed in Z-P-R ternary complex, while Z-P-L ternary complex remained the spherical structure. These findings would provide new insights into the development of novel delivery system and expand the options, when zein-based complexes were utilized under different environment conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biomimicry of surfactant protein C.

Science.gov (United States)

Brown, Nathan J; Johansson, Jan; Barron, Annelise E

2008-10-01

Since the widespread use of exogenous lung surfactant to treat neonatal respiratory distress syndrome, premature infant survival and respiratory morbidity have dramatically improved. Despite the effectiveness of the animal-derived surfactant preparations, there still remain some concerns and difficulties associated with their use. This has prompted investigation into the creation of synthetic surfactant preparations. However, to date, no clinically used synthetic formulation is as effective as the natural material. This is largely because the previous synthetic formulations lacked analogues of the hydrophobic proteins of the lung surfactant system, SP-B and SP-C, which are critical functional constituents. As a result, recent investigation has turned toward the development of a new generation of synthetic, biomimetic surfactants that contain synthetic phospholipids along with a mimic of the hydrophobic protein portion of lung surfactant. In this Account, we detail our efforts in creating accurate mimics of SP-C for use in a synthetic surfactant replacement therapy. Despite SP-C's seemingly simple structure, the predominantly helical protein is extraordinarily challenging to work with given its extreme hydrophobicity and structural instability, which greatly complicates the creation of an effective SP-C analogue. Drawing inspiration from Nature, two promising biomimetic approaches have led to the creation of rationally designed biopolymers that recapitulate many of SP-C's molecular features. The first approach utilizes detailed SP-C structure-activity relationships and amino acid folding propensities to create a peptide-based analogue, SP-C33. In SP-C33, the problematic and metastable polyvaline helix is replaced with a structurally stable polyleucine helix and includes a well-placed positive charge to prevent aggregation. SP-C33 is structurally stable and eliminates the association propensity of the native protein. The second approach follows the same design

Pulmonary haptoglobin (pHp) is part of the surfactant system in the human lung.

Science.gov (United States)

Abdullah, Mahdi; Goldmann, Torsten

2012-11-20

Since the existence of pHp was demonstrated, it has been shown that this molecule and its receptor CD163 are regulated by different stimuli. Furthermore, a comparably fast secretion of pHp was described as well as the immuno-stimulatory effects. The intention of this study was to elucidate the role of pHp in the human lungs further. Here we show, by means of confocal microscopy and immune-electron-microscopy, a clear co-localization of pHp with surfactant protein-B in lamellar bodies of alveolar epithelial cells type II. These results are underlined by immunohistochemical stainings in differently fixed human lung tissues, which show pHp in vesicular and released form. The images of the released form resemble the intended position of surfactant in the human alveolus. pHp is secreted by Alveolar epithelial cells type II as previously shown. Moreover, pHp is co-localized with Surfactant protein-B. We conclude that the presented data shows that pHp is a native part of the surfactant system in the human lung. http://www.diagnosticpathology.diagnomx.eu/vs/2563584738239912.

Remediation using trace element humate surfactant

Energy Technology Data Exchange (ETDEWEB)

Riddle, Catherine Lynn; Taylor, Steven Cheney; Bruhn, Debra Fox

2016-08-30

A method of remediation at a remediation site having one or more undesirable conditions in which one or more soil characteristics, preferably soil pH and/or elemental concentrations, are measured at a remediation site. A trace element humate surfactant composition is prepared comprising a humate solution, element solution and at least one surfactant. The prepared trace element humate surfactant composition is then dispensed onto the remediation site whereby the trace element humate surfactant composition will reduce the amount of undesirable compounds by promoting growth of native species activity. By promoting native species activity, remediation occurs quickly and environmental impact is minimal.

Synthesis of carbohydrate-based surfactants

Energy Technology Data Exchange (ETDEWEB)

Pemberton, Jeanne E.; Polt, Robin L.; Maier, Raina M.

2016-11-22

The present invention provides carbohydrate-based surfactants and methods for producing the same. Methods for producing carbohydrate-based surfactants include using a glycosylation promoter to link a carbohydrate or its derivative to a hydrophobic compound.

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...

Small angle neutron scattering study of doxorubicin–surfactant ...

Indian Academy of Sciences (India)

The binding affinity of doxorubicin within the micelle carrier is enhanced through complex formation of drug and anionic surfactant, aerosol OT (AOT). Electrostatic binding of doxorubicin with negatively charged surfactants leads to the formation of hydrophobic drug–surfactant complexes. Surfactant-induced partitioning of ...

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.

DNA compaction by azobenzene-containing surfactant

International Nuclear Information System (INIS)

Zakrevskyy, Yuriy; Kopyshev, Alexey; Lomadze, Nino; Santer, Svetlana; Morozova, Elena; Lysyakova, Ludmila; Kasyanenko, Nina

2011-01-01

We report on the interaction of cationic azobenzene-containing surfactant with DNA investigated by absorption and fluorescence spectroscopy, dynamic light scattering, and atomic force microscopy. The properties of the surfactant can be controlled with light by reversible switching of the azobenzene unit, incorporated into the surfactant tail, between a hydrophobic trans (visible irradiation) and a hydrophilic cis (UV irradiation) configuration. The influence of the trans-cis isomerization of the azobenzene on the compaction process of DNA molecules and the role of both isomers in the formation and colloidal stability of DNA-surfactant complexes is discussed. It is shown that the trans isomer plays a major role in the DNA compaction process. The influence of the cis isomer on the DNA coil configuration is rather small. The construction of a phase diagram of the DNA concentration versus surfactant/DNA charge ratio allows distancing between three major phases: colloidally stable and unstable compacted globules, and extended coil conformation. There is a critical concentration of DNA above which the compacted globules can be hindered from aggregation and precipitation by adding an appropriate amount of the surfactant in the trans configuration. This is because of the compensation of hydrophobicity of the globules with an increasing amount of the surfactant. Below the critical DNA concentration, the compacted globules are colloidally stable and can be reversibly transferred with light to an extended coil state.

Isolation, purification, and partial characterization of a membrane-bound Cl-/HCO3--activated ATPase complex from rat brain with sensitivity to GABAAergic ligands.

Science.gov (United States)

Menzikov, Sergey A

2017-02-07

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

Viscosity of the oil-in-water Pickering emulsion stabilized by surfactant-polymer and nanoparticle-surfactant-polymer system

Science.gov (United States)

Sharma, Tushar; Kumar, G. Suresh; Chon, Bo Hyun; Sangwai, Jitendra S.

2014-11-01

Information on the viscosity of Pickering emulsion is required for their successful application in upstream oil and gas industry to understand their stability at extreme environment. In this work, a novel formulation of oil-in-water (o/w) Pickering emulsion stabilized using nanoparticle-surfactant-polymer (polyacrylamide) system as formulated in our earlier work (Sharma et al., Journal of Industrial and Engineering Chemistry, 2014) is investigated for rheological stability at high pressure and high temperature (HPHT) conditions using a controlled-strain rheometer. The nanoparticle (SiO2 and clay) concentration is varied from 1.0 to 5.0 wt%. The results are compared with the rheological behavior of simple o/w emulsion stabilized by surfactant-polymer system. Both the emulsions exhibit non-Newtonian shear thinning behavior. A positive shift in this behavior is observed for surfactant-polymer stabilized emulsion at high pressure conditions. Yield stress is observed to increase with pressure for surfactant-polymer emulsion. In addition, increase in temperature has an adverse effect on the viscosity of emulsion stabilized by surfactant-polymer system. In case of nanoparticle-surfactant-polymer stabilized o/w emulsion system, the viscosity and yield stress are predominantly constant for varying pressure and temperature conditions. The viscosity data for both o/w emulsion systems are fitted by the Herschel-Bulkley model and found to be satisfactory. In general, the study indicates that the Pickering emulsion stabilized by nanoparticle-surfactant-polymer system shows improved and stable rheological properties as compared to conventional emulsion stabilized by surfactant-polymer system indicating their successful application for HPHT environment in upstream oil and gas industry.

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Science.gov (United States)

Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Directory of Open Access Journals (Sweden)

Kazi Md Kamrul Huda

Full Text Available Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible

Arcuate Na+,K+-ATPase senses systemic energy states and regulates feeding behavior through glucose-inhibited neurons.

Science.gov (United States)

Kurita, Hideharu; Xu, Kai Y; Maejima, Yuko; Nakata, Masanori; Dezaki, Katsuya; Santoso, Putra; Yang, Yifei; Arai, Takeshi; Gantulga, Darambazar; Muroya, Shinji; Lefor, Alan K; Kakei, Masafumi; Watanabe, Eiju; Yada, Toshihiko

2015-08-15

Feeding is regulated by perception in the hypothalamus, particularly the first-order arcuate nucleus (ARC) neurons, of the body's energy state. However, the cellular device for converting energy states to the activity of critical neurons in ARC is less defined. We here show that Na(+),K(+)-ATPase (NKA) in ARC senses energy states to regulate feeding. Fasting-induced systemic ghrelin rise and glucose lowering reduced ATP-hydrolyzing activity of NKA and its substrate ATP level, respectively, preferentially in ARC. Lowering glucose concentration (LG), which mimics fasting, decreased intracellular NAD(P)H and increased Na(+) concentration in single ARC neurons that subsequently exhibited [Ca(2+)]i responses to LG, showing that they were glucose-inhibited (GI) neurons. Third ventricular injection of the NKA inhibitor ouabain induced c-Fos expression in agouti-related protein (AgRP) neurons in ARC and evoked neuropeptide Y (NPY)-dependent feeding. When injected focally into ARC, ouabain stimulated feeding and mRNA expressions for NPY and AgRP. Ouabain increased [Ca(2+)]i in single NPY/AgRP neurons with greater amplitude than in proopiomelanocortin neurons in ARC. Conversely, the specific NKA activator SSA412 suppressed fasting-induced feeding and LG-induced [Ca(2+)]i increases in ARC GI neurons. NPY/AgRP neurons highly expressed NKAα3, whose knockdown impaired feeding behavior. These results demonstrate that fasting, via ghrelin rise and LG, suppresses NKA enzyme/pump activity in ARC and thereby promotes the activation of GI neurons and NPY/AgRP-dependent feeding. This study identifies ARC NKA as a hypothalamic sensor and converter of metabolic states to key neuronal activity and feeding behaviour, providing a new target to treat hyperphagic obesity and diabetes. Copyright © 2015 the American Physiological Society.

Low pH-induced changes of antioxidant enzyme and ATPase activities in the roots of rice (Oryza sativa L. seedlings.

Directory of Open Access Journals (Sweden)

Yi-Kai Zhang

Full Text Available Soil acidification is the main problem in the current rice production. Here, the effects of low pH on the root growth, reactive oxygen species metabolism, plasma membrane functions, and the transcript levels of the related genes were investigated in rice seedlings (Oryza sativa L. in a hydroponic system at pH 3.5, 4.5, and 5.5. There were two hybrid rice cultivars in this trial, including Yongyou 12 (YY12, a japonica hybrid and Zhongzheyou 1 (ZZY1, an indica hybrid. Higher H+ activity markedly decreased root length, the proportion of fine roots, and dry matter production, but induced a significant accumulation of hydrogen peroxide (H2O2, and led to serious lipid peroxidation in the roots of the two varieties. The transcript levels of copper/zinc superoxide dismutase 1 (Cu/Zn SOD1, copper/zinc superoxide dismutase 2 (Cu/Zn SOD2, catalase A (CATA and catalase B (CATB genes in YY12 and ZZY1 roots were significantly down-regulated after low pH exposure for two weeks. Meanwhile, a significant decrease was observed in the expression of the P-type Ca2+-ATPases in roots at pH 3.5. The activities of antioxidant enzymes (SOD, CAT and plasma membrane (PM Ca2+-ATPase in the two varieties were dramatically inhibited by strong rhizosphere acidification. However, the expression levels of ascorbate peroxidase 1 (APX1 and PM H+-ATPase isoform 7 were up-regulated under H+ stress compared with the control. Significantly higher activities of APX and PM H+-ATPase could contribute to the adaptation of rice roots to low pH.

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.

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...

Binding of cationic surfactants to humic substances

NARCIS (Netherlands)

Ishiguro, M.; Tan, W.; Koopal, L.K.

2007-01-01

Commercial surfactants are introduced into the environment either through waste products or site-specific contamination. The amphiphilic nature of both surfactants and humic substances (HS) leads to their mutual attraction especially when surfactant and HS are oppositely charged. Binding of the

Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast

Science.gov (United States)

Sze, H.; Liang, F.; Hwang, I.; Curran, A. C.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The spatial and temporal regulation of calcium concentration in plant cells depends on the coordinate activities of channels and active transporters located on different organelles and membranes. Several Ca2+ pumps have been identified and characterized by functional expression of plant genes in a yeast mutant (K616). This expression system has opened the way to a genetic and biochemical characterization of the regulatory and catalytic features of diverse Ca2+ pumps. Plant Ca(2+)-ATPases fall into two major types: AtECA1 represents one of four or more members of the type IIA (ER-type) Ca(2+)-ATPases in Arabidopsis, and AtACA2 is one of seven or more members of the type IIB (PM-type) Ca(2+)-ATPases that are regulated by a novel amino terminal domain. Type IIB pumps are widely distributed on membranes, including the PM (plasma membrane), vacuole, and ER (endoplasmic reticulum). The regulatory domain serves multiple functions, including autoinhibition, calmodulin binding, and sites for modification by phosphorylation. This domain, however, is considerably diverse among several type IIB ATPases, suggesting that the pumps are differentially regulated. Understanding of Ca2+ transporters at the molecular level is providing insights into their roles in signaling networks and in regulating fundamental processes of cell biology.

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.

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.

Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study.

Directory of Open Access Journals (Sweden)

Silvia Petrezsélyová

Full Text Available Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.

Surfactants tailored by the class Actinobacteria

Science.gov (United States)

Kügler, Johannes H.; Le Roes-Hill, Marilize; Syldatk, Christoph; Hausmann, Rudolf

2015-01-01

Globally the change towards the establishment of a bio-based economy has resulted in an increased need for bio-based applications. This, in turn, has served as a driving force for the discovery and application of novel biosurfactants. The class Actinobacteria represents a vast group of microorganisms with the ability to produce a diverse range of secondary metabolites, including surfactants. Understanding the extensive nature of the biosurfactants produced by actinobacterial strains can assist in finding novel biosurfactants with new potential applications. This review therefore presents a comprehensive overview of the knowledge available on actinobacterial surfactants, the chemical structures that have been completely or partly elucidated, as well as the identity of the biosurfactant-producing strains. Producer strains of not yet elucidated compounds are discussed, as well as the original habitats of all the producer strains, which seems to indicate that biosurfactant production is environmentally driven. Methodology applied in the isolation, purification and structural elucidation of the different types of surface active compounds, as well as surfactant activity tests, are also discussed. Overall, actinobacterial surfactants can be summarized to include the dominantly occurring trehalose-comprising surfactants, other non-trehalose containing glycolipids, lipopeptides and the more rare actinobacterial surfactants. The lack of structural information on a large proportion of actinobacterial surfactants should be considered as a driving force to further explore the abundance and diversity of these compounds. This would allow for a better understanding of actinobacterial surface active compounds and their potential for biotechnological application. PMID:25852670

Surfactants tailored by the class Actinobacteria

Directory of Open Access Journals (Sweden)

Johannes H Kügler

2015-03-01

Full Text Available Gloablly, the drive towards the establishment of a bio-based economy has resulted in an increased need for bio-based applications. This, in turn, has served as a driving force for the discovery and application of novel biosurfactants. The class Actinobacteria represents a vast group of microorganisms with the ability to produce a diverse range of secondary metabolites, including surfactants. Understanding the extensive nature of the biosurfactants produced by actinobacterial strains can assist in finding novel biosurfactants with new potential applications. This review therefore presents a comprehensive overview of the knowledge available on actinobacterial surfactants, the chemical structures that have been completely or partly elucidated, as well as the identity of the biosurfactant-producing strains. Producer strains of not yet elucidated compounds are discussed, as well as the original habitats of all the producer strains, which seems to indicate that biosurfactant production is environmentally driven. Methodology applied in the isolation, purification and structural elucidation of the different types of surface active compounds, as well as surfactant activity tests, are also discussed. Overall, actinobacterial surfactants can be summarized to include the dominantly occurring trehalose-comprising surfactants, other non-trehalose containing glycolipids, lipopeptides and the more rare actinobacterial surfactants. The lack of structural information on a large proportion of actinobacterial surfactants should be considered as a driving force to further explore the abundance and diversity of these compounds. This would allow for a better understanding of actinobacterial surface active compounds and their potential for biotechnological application.

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...

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

The potential of fluorinated surfactants in membrane biochemistry.

Science.gov (United States)

Shepherd, F H; Holzenburg, A

1995-01-01

Detergents are important reagents in membrane biochemistry. Since each membrane system studied places different demands on the detergent in terms of desirous physicochemical properties, detergents new to biochemistry must continuously be sought. Ammonium perfluorooctanoate (APFO) was investigated, as representative of fluorinated surfactants, in terms of its suitability as a "biological detergent." It did not interfere with the Markwell modification of the Lowry procedure at detergent concentrations of up to 2% (w/v). Critical micellization concentration (cmc) values (0.013-0.0275 M) for this detergent were determined in a number of buffers of biological interest. It was demonstrated that the detergent can be removed by dialysis, albeit slowly. This slow removal may be particularly useful for reconstitution/crystallization studies. Solubilization studies on several membrane systems containing the proteins listed (the major protein of the membrane sector of the vacuolar H(+)-ATPase (16 kDa protein); photosystem II; equine herpes virus (EHV) envelope proteins) indicate that it is a potent solubilizing agent, likely to enhance the yield in cases where solubilization has already been demonstrated, and, in other cases, to solubilize proteins formerly recalcitrant to solubilization. The removal of APFO from solubilized 16-kDa protein by means of Extracti-Gel D resin as a means of exchanging detergents quickly and with a minimum requirement for second detergent was investigated.

In vitro surfactant structure-toxicity relationships: implications for surfactant use in sexually transmitted infection prophylaxis and contraception.

Directory of Open Access Journals (Sweden)

Ângela S Inácio

Full Text Available BACKGROUND: The need for woman-controlled, cheap, safe, effective, easy-to-use and easy-to-store topical applications for prophylaxis against sexually transmitted infections (STIs makes surfactant-containing formulations an interesting option that requires a more fundamental knowledge concerning surfactant toxicology and structure-activity relationships. METHODOLOGY/PRINCIPAL FINDINGS: We report in vitro effects of surfactant concentration, exposure time and structure on the viability of mammalian cell types typically encountered in the vagina, namely, fully polarized and confluent epithelial cells, confluent but non-polarized epithelial-like cells, dendritic cells, and human sperm. Representatives of the different families of commercially available surfactants--nonionic (Triton X-100 and monolaurin, zwitterionic (DDPS, anionic (SDS, and cationic (C(nTAB (n = 10 to 16, C(12PB, and C(12BZK--were examined. Triton X-100, monolaurin, DDPS and SDS were toxic to all cell types at concentrations around their critical micelle concentration (CMC suggesting a non-selective mode of action involving cell membrane destabilization and/or destruction. All cationic surfactants were toxic at concentrations far below their CMC and showed significant differences in their toxicity toward polarized as compared with non-polarized cells. Their toxicity was also dependent on the chemical nature of the polar head group. Our results suggest an intracellular locus of action for cationic surfactants and show that their structure-activity relationships could be profitably exploited for STI prophylaxis in vaginal gel formulations. The therapeutic indices comparing polarized epithelial cell toxicity to sperm toxicity for all surfactants examined, except C(12PB and C(12BZK, does not justify their use as contraceptive agents. C(12PB and C(12BZK are shown to have a narrow therapeutic index recommending caution in their use in contraceptive formulations. CONCLUSIONS

Next Generation Surfactants for Improved Chemical Flooding Technology

Energy Technology Data Exchange (ETDEWEB)

Laura Wesson; Prapas Lohateeraparp; Jeffrey Harwell; Bor-Jier Shiau

2012-05-31

The principle objective of this project was to characterize and test current and next generation high performance surfactants for improved chemical flooding technology, focused on reservoirs in the Pennsylvanian-aged (Penn) sands. In order to meet this objective the characteristic curvatures (Cc) of twenty-eight anionic surfactants selected for evaluation for use in chemical flooding formulations were determined. The Cc values ranged from -6.90 to 2.55 with the majority having negative values. Crude oil samples from nine Penn sand reservoirs were analyzed for several properties pertinent to surfactant formulation for EOR application. These properties included equivalent alkane carbon numbers, total acid numbers, and viscosity. The brine samples from these same reservoirs were analyzed for several cations and for total dissolved solids. Surfactant formulations were successfully developed for eight reservoirs by the end of the project period. These formulations were comprised of a tertiary mixture of anionic surfactants. The identities of these surfactants are considered proprietary, but suffice to say the surfactants in each mixture were comprised of varying chemical structures. In addition to the successful development of surfactant formulations for EOR, there were also two successful single-well field tests conducted. There are many aspects that must be considered in the development and implementation of effective surfactant formulations. Taking into account these other aspects, there were four additional studies conducted during this project. These studies focused on the effect of the stability of surfactant formulations in the presence of polymers with an associated examination of polymer rheology, the effect of the presence of iron complexes in the brine on surfactant stability, the potential use of sacrificial agents in order to minimize the loss of surfactant to adsorption, and the effect of electrolytes on surfactant adsorption. In these last four studies

Hypoxia Stress Modifies Na+/K+-ATPase, H+/K+-ATPase, [Formula: see text], and nkaα1 Isoform Expression in the Brain of Immune-Challenged Air-Breathing Fish.

Science.gov (United States)

Peter, Mc Subhash; Simi, Satheesan

2017-01-01

Fishes are equipped to sense stressful stimuli and are able to respond to environmental stressor such as hypoxia with varying pattern of stress response. The functional attributes of brain to hypoxia stress in relation to ion transport and its interaction during immune challenge have not yet delineated in fish. We, therefore, explored the pattern of ion transporter functions and messenger RNA (mRNA) expression of α1-subunit isoforms of Na + /K + -ATPase (NKA) in the brain segments, namely, prosencephalon (PC), mesencephalon (MC), and metencephalon (MeC) in an obligate air-breathing fish exposed either to hypoxia stress (30 minutes forced immersion in water) or challenged with zymosan treatment (25-200 ng g -1 for 24 hours) or both. Zymosan that produced nonspecific immune responses evoked differential regulation of NKA, H + /K + -ATPase (HKA), and [Formula: see text] (NNA) in the varied brain segments. On the contrary, hypoxia stress that demanded activation of NKA in PC and MeC showed a reversed NKA activity pattern in MeC of immune-challenged fish. A compromised HKA and NNA regulation during hypoxia stress was found in immune-challenged fish, indicating the role of these brain ion transporters to hypoxia stress and immune challenges. The differential mRNA expression of α1-subunit isoforms of NKA, nkaα1a , nkaα1b , and nkaα1c , in hypoxia-stressed brain showed a shift in its expression pattern during hypoxia stress-immune interaction in PC and MC. Evidence is thus presented for the first time that ion transporters such as HKA and NNA along with NKA act as functional brain markers which respond differentially to both hypoxia stress and immune challenges. Taken together, the data further provide evidence for a differential Na + , K + , H + , and [Formula: see text] ion signaling that exists in brain neuronal clusters during hypoxia stress-immune interaction as a result of modified regulations of NKA, HKA, and NNA transporter functions and nkaα1 isoform

The Molecular Era of Surfactant Biology

OpenAIRE

Whitsett, Jeffrey A.

2014-01-01

Advances in the physiology, biochemistry, molecular and cell biology of the pulmonary surfactant system transformed the clinical care and outcome of preterm infants with respiratory distress syndrome. The molecular era of surfactant biology provided genetic insights into the pathogenesis of pulmonary disorders, previously termed “idiopathic” that affect newborn infants, children and adults. Knowledge related to the structure and function of the surfactant proteins and their roles in alveolar ...

Nanoparticle-enabled delivery of surfactants in porous media.

Science.gov (United States)

Nourafkan, Ehsan; Hu, Zhongliang; Wen, Dongsheng

2018-06-01

The adsorption of surfactants on the reservoir rocks surface is a serious issue in many energy and environment related areas. Learning from the concept of drug delivery in the nano-medicine field, this work proposes and validates the concept of using nanoparticles to deliver a mixture of surfactants into a porous medium. TiO 2 nanoparticles (NPs) are used as carriers for a blend of surfactants mixtures including anionic alkyl aryl sulfonic acid (AAS) and nonionic alcohol ethoxylated (EA) at the optimum salinity and composition conditions. The transport of NPs through a core sample of crushed sandstone grains and the adsorption of surfactants are evaluated. By using TiO 2 NPs, the adsorption of surfactant molecules can be significantly reduced, i.e. half of the initial adsorption value. The level of surfactant adsorption reduction is related to the NPs transport capability through the porous medium. An application study shows that comparing to surfactant flooding alone, the total oil recovery can be increased by 7.81% of original oil in place (OOIP) by using nanoparticle bonded surfactants. Such work shows the promise of NP as an effective surfactant carrier for sandstone reservoirs, which could have many potential applications in enhanced oil recovery (EOR) and environmental remediation. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of rSP-C surfactant with natural and synthetic surfactants after late treatment in a rat model of the acute respiratory distress syndrome

Science.gov (United States)

Häfner, Dietrich; Germann, Paul-Georg; Hauschke, Dieter

1998-01-01

In a previous paper we showed that an SP-C containing surfactant preparation has similar activity as bovine-derived surfactants in a rat lung lavage model of the adult respiratory distress syndrome. In this study surfactant was given ten minutes after the last lavage (early treatment). In the present investigation we were interested how different surfactant preparations behave when they are administered 1 h after the last lavage (late treatment). Four protein containing surfactants (rSP-C surfactant, bLES, Infasurf and Survanta) were compared with three protein-free surfactants (ALEC, Exosurf and the phospholipid (PL) mixture of the rSP-C surfactant termed PL surfactant) with respect to their ability to improve gas exchange in this more stringent model when surfactant is given one hour after the last lavage. For better comparison of the surfactants the doses were related to phospholipids. The surfactants were given at doses of 25, 50 and 100 mg kg−1 body weight. The surfactants were compared to an untreated control group that was only ventilated for the whole experimental period. Tracheotomized rats (8–12 per dose and surfactant) were pressure-controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths min−1, inspiration expiration ratio of 1 : 2, peak inspiratory pressure of 28 cmH2O at positive endexpiratory pressure (PEEP) of 8 cmH2O. Animals were ventilated for one hour after the last lavage and thereafter the surfactants were intratracheally instilled. During the whole experimental period the ventilation was not changed. Partial arterial oxygen pressures (PaO2, mmHg) at 30 min and 120 min after treatment were used for statistical comparison. All protein containing surfactants caused a dose-dependent increase of the reduced PaO2 values at 30 min after treatment. The protein-free surfactants showed only weak dose-dependent increase in PaO2 values at this time. This difference between the

Surfactant modified clays’ consistency limits and contact angles

Directory of Open Access Journals (Sweden)

S Akbulut

2012-07-01

Full Text Available This study was aimed at preparing a surfactant modified clay (SMC and researching the effect of surfactants on clays' contact angles and consistency limits; clay was thus modified by surfactants formodifying their engineering properties. Seven surfactants (trimethylglycine, hydroxyethylcellulose  octyl phenol ethoxylate, linear alkylbenzene sulfonic acid, sodium lauryl ether sulfate, cetyl trimethylammonium chloride and quaternised ethoxylated fatty amine were used as surfactants in this study. The experimental results indicated that SMC consistency limits (liquid and plastic limits changedsignificantly compared to those of natural clay. Plasticity index and liquid limit (PI-LL values representing soil class approached the A-line when zwitterion, nonionic, and anionic surfactant percentageincreased. However, cationic SMC became transformed from CH (high plasticity clay to MH (high plasticity silt class soils, according to the unified soil classification system (USCS. Clay modifiedwith cationic and anionic surfactants gave higher and lower contact angles than natural clay, respectively.

Probing dynamics and mechanism of exchange process of quaternary ammonium dimeric surfactants, 14-s-14, in the presence of conventional surfactants.

Science.gov (United States)

Liu, Jun; Jiang, Yan; Chen, Hong; Mao, Shi Zhen; Du, You Ru; Liu, Mai Li

2012-12-27

In this Article, we investigated effects of different types of conventional surfactants on exchange dynamics of quaternary ammonium dimeric surfactants, with chemical formula C(14)H(29)N(+)(CH(3))(2)- (CH(2))(s)-N(+)(CH(3))(2)C(14)H(29)·2Br(-), or 14-s-14 for short. Two nonionic surfactants, TritonX-100 (TX-100) and polyethylene glycol (23) laurylether (Brij-35), and one cationic surfactant, n-tetradecyltrimethyl ammonium bromide (TTAB), and one ionic surfactant, sodium dodecyl sulfate (SDS) were chosen as typical conventional surfactants. Exchange rates of 14-s-14 (s = 2, 3, and 4) between the micelle form and monomer in solution were detected by two NMR methods: one-dimensional (1D) line shape analysis and two-dimensional (2D) exchange spectroscopy (EXSY). Results show that the nonionic surfactants (TX-100 and Brij-35), the cationic surfactant (TTAB), and the ionic surfactant (SDS) respectively accelerated, barely influenced, and slowed the exchange rate of 14-s-14. The effect mechanism was investigated by the self-diffusion experiment, relaxation time measurements (T(2)/T(1)), the fluorescence experiment (I(1)/I(3)) and observed chemical shift variations. Results reveal that, nonionic conventional surfactants (TX-100 and Brij-35) loosened the molecule arrangement and decreased hydrophobic interactions in the micelle, and thus accelerated the exchange rate of 14-s-14. The cationic conventional surfactant (TTAB) barely changed the molecule arrangement and thus barely influenced the exchange rate of 14-s-14. The ionic conventional surfactant (SDS) introduced the electrostatic attraction effect, tightened the molecule arrangement, and increased hydrophobic interactions in the micelle, and thus slowed down the exchange rate of 14-s-14. Additionally, the two-step exchange mechanism of 14-s-14 in the mixed solution was revealed through interesting variation tendencies of exchange rates of 14-s-14.

C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.

Directory of Open Access Journals (Sweden)

Dana Galuska

Full Text Available Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86Rb(+ uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM. DNA binding activity was determined by electrical mobility shift assay (EMSA. Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1-subunit protein expression, accompanied with increase in (86Rb(+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6, concomitant with Na,K-ATPase α(1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C

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.

Effects of Surfactant on Geotechnical Characteristics of Silty Soil

International Nuclear Information System (INIS)

Rahman, Z.A.; Sahibin, A.R.; Lihan, T.; Idris, W.M.R.; Sakina, M.

2013-01-01

Surfactants are often used as a cleaning agent for restoration of oil-contaminated soil. However the effect of surfactant on the geotechnical properties of soil is not clearly understood. In this study, the effects of surfactant on silty soil were investigated for consistency index, compaction, permeability and shear strength. Sodium dodecyl sulfate (SDS) was used in this study to prepare the surfactant-treated soil. Our results showed that the soil with added surfactant exhibited a decrease in liquid and plastic limit values. Maximum dry densities increased and optimum moisture contents decreased as contents of added surfactant were increased. The presence of surfactant assists the soil to achieve maximum density at lower water content. The addition of surfactant decreased the permeability of soil from 6.29 x 10 -4 to 1.15 x 10 -4 ms -1 . The shear strength of soil with added surfactant was examined using the undrained unconsolidated triaxial tests. The results showed that the undrained shear strength, Cu was significantly affected, decreased from 319 kPa to 50 kPa for soil with 20 % of added surfactant. The results of this study showed that the presence of surfactant in soil can modify the mechanical behaviour of the soil. (author)

Surfactant-enhanced control of track-etch pore morphology

International Nuclear Information System (INIS)

Apel', P.Yu.; Blonskaya, I.V.; Didyk, A.Yu.; Dmitriev, S.N.; Orelovich, O.L.; Samojlova, L.I.; Vutsadakis, V.A.; Root, D.

2000-01-01

The influence of surfactants on the process of chemical development of ion tracks in polymers is studied. Based on the experimental data, a mechanism of the surfactant effect on the track-etch pore morphology is proposed. In the beginning of etching the surfactant is adsorbed on the surface and creates a layer that is quasi-solid and partially protects the surface from the etching agent. However, some etchant molecules diffuse through the barrier and react with the polymer surface. This results in the formation of a small hole at the entrance to the ion track. After the hole has attained a few annometers in diameter, the surfactant molecules penetrate into the track and cover its walls. Further diffusion of the surfactant into the growing pore is hindered. The adsorbed surfactant layer is not permeable for large molecules. In contrast, small alkali molecules and water molecules diffuse into the track and provide the etching process enlarging the pore. At this stage the transport of the surfactant into the pore channel can proceed only due to the lateral diffusion in the adsorbed layer. The volume inside the pore is free of surfactant molecules and grows at a higher rate than pore entrance. After a more prolonged etching the bottle-like (or 'cigar-like') pore channels are formed. The bottle-like shape of the pore channels depends on the etching conditions such as alkali and surfactant concentration, temperature, and type of the surfactant. The use of surfactants enables one to produce track-etch membranes with improved flow rate characteristics compared with those having cylindrical pores with the same nominal pore diameters

Open lung ventilation preserves the response to delayed surfactant treatment in surfactant-deficient newborn piglets

NARCIS (Netherlands)

van Veenendaal, Mariëtte B.; van Kaam, Anton H.; Haitsma, Jack J.; Lutter, René; Lachmann, Burkhard

2006-01-01

OBJECTIVE: Delayed surfactant treatment (>2 hrs after birth) is less effective than early treatment in conventionally ventilated preterm infants with respiratory distress syndrome. The objective of this study was to evaluate if this time-dependent efficacy of surfactant treatment is also present

Influence of surfactants in forced dynamic dewetting.

Science.gov (United States)

Henrich, Franziska; Fell, Daniela; Truszkowska, Dorota; Weirich, Marcel; Anyfantakis, Manos; Nguyen, Thi-Huong; Wagner, Manfred; Auernhammer, Günter K; Butt, Hans-Jürgen

2016-09-20

In this work we show that the forced dynamic dewetting of surfactant solutions depends sensitively on the surfactant concentration. To measure this effect, a hydrophobic rotating cylinder was horizontally half immersed in aqueous surfactant solutions. Dynamic contact angles were measured optically by extrapolating the contour of the meniscus to the contact line. Anionic (sodium 1-decanesulfonate, S-1DeS), cationic (cetyl trimethylammonium bromide, CTAB) and nonionic surfactants (C 4 E 1 , C 8 E 3 and C 12 E 5 ) with critical micelle concentrations (CMCs) spanning four orders of magnitude were used. The receding contact angle in water decreased with increasing velocity. This decrease was strongly enhanced when adding surfactant, even at surfactant concentrations of 10% of the critical micelle concentration. Plots of the receding contact angle-versus-velocity almost superimpose when being plotted at the same relative concentration (concentration/CMC). Thus the rescaled concentration is the dominating property for dynamic dewetting. The charge of the surfactants did not play a role, thus excluding electrostatic effects. The change in contact angle can be interpreted by local surface tension gradients, i.e. Marangoni stresses, close to the three-phase contact line. The decrease of dynamic contact angles with velocity follows two regimes. Despite the existence of Marangoni stresses close to the contact line, for a dewetting velocity above 1-10 mm s -1 the hydrodynamic theory is able to describe the experimental results for all surfactant concentrations. At slower velocities an additional steep decrease of the contact angle with velocity was observed. Particle tracking velocimetry showed that the flow profiles do not differ with and without surfactant on a scales >100 μm.

Dimeric Surfactants: Promising Ingredients of Cosmetics and Toiletries

Directory of Open Access Journals (Sweden)

Naveen Kumar

2013-11-01

Full Text Available Surfactants are an essential ingredient for cosmetic, toiletries and personal care products for enhancing their performance. Dimeric surfactants demonstrate superiority compared to conventional surfactants in all areas of application. Dimeric surfactants are extremely promising for utilization in various cosmetic formulations viz. shampoo, lotions, creams, conditioners etc. These surfactants possess extremely unique surface properties viz. lower surface tension, unique micellization, low critical micelle concentration (CMC and antimicrobial activity, higher solubilization etc. Dimerics enhance the performances of cosmetics in an extraordinary manner and provide eco-friendly preparations for human epidermis.

Structural study of surfactant-dependent interaction with protein

Energy Technology Data Exchange (ETDEWEB)

Mehan, Sumit; Aswal, Vinod K., E-mail: vkaswal@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kohlbrecher, Joachim [Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen (Switzerland)

2015-06-24

Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

Effect of double-tailed surfactant architecture on the conformation, self-assembly, and processing in polypeptide-surfactant complexes.

Science.gov (United States)

Junnila, Susanna; Hanski, Sirkku; Oakley, Richard J; Nummelin, Sami; Ruokolainen, Janne; Faul, Charl F J; Ikkala, Olli

2009-10-12

This work describes the solid-state conformational and structural properties of self-assembled polypeptide-surfactant complexes with double-tailed surfactants. Poly(L-lysine) was complexed with three dialkyl esters of phosphoric acid (i.e., phosphodiester surfactants), where the surfactant tail branching and length was varied to tune the supramolecular architecture in a facile way. After complexation with the branched surfactant bis(2-ethylhexyl) phosphate in an aqueous solution, the polypeptide chains adopted an alpha-helical conformation. These rod-like helices self-assembled into cylindrical phases with the amorphous alkyl tails pointing outward. In complexes with dioctyl phosphate and didodecyl phosphate, which have two linear n-octyl or n-dodecyl tails, respectively, the polypeptide formed antiparallel beta-sheets separated by alkyl layers, resulting in well-ordered lamellar self-assemblies. By heating, it was possible to trigger a partial opening of the beta-sheets and disruption of the lamellar phase. After repeated heating/cooling, all of these complexes also showed a glass transition between 37 and 50 degrees C. Organic solvent treatment and plasticization by overstoichiometric amount of surfactant led to structure modification in poly(L-lysine)-dioctyl phosphate complexes, PLL(diC8)(x) (x = 1.0-3.0). Here, the alpha-helical PLL is surrounded by the surfactants and these bottle-brush-like chains self-assemble in a hexagonal cylindrical morphology. As x is increased, the materials are clearly plasticized and the degree of ordering is improved: The stiff alpha-helical backbones in a softened surfactant matrix give rise to thermotropic liquid-crystalline phases. The complexes were examined by Fourier transform infrared spectroscopy, small- and wide-angle X-ray scattering, transmission electron microscopy, differential scanning calorimetry, polarized optical microscopy, and circular dichroism.

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

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.

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca"2"+-ATPase (SERCA) activity

International Nuclear Information System (INIS)

Benaim, Gustavo; Pimentel, Adriana A.; Felibertt, Pimali; Mayora, Adriana; Colman, Laura; Sojo, Felipe; Rojas, Héctor; De Sanctis, Juan B.

2016-01-01

The increase in the intracellular Ca"2"+ concentration ([Ca"2"+]_i) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca"2"+]_i by inhibiting the sarco(endo)plasmic reticulum Ca"2"+-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca"2"+ pump. The results showed that addition of sphingosine produced a release of Ca"2"+ from the endoplasmic reticulum followed by a Ca"2"+ entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca"2"+]_I in mammalian cells.

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.

The Biophysical Function of Pulmonary Surfactant

OpenAIRE

Rugonyi, Sandra; Biswas, Samares C.; Hall, Stephen B.

2008-01-01

Pulmonary surfactant lowers surface tension in the lungs. Physiological studies indicate two key aspects of this function: that the surfactant film forms rapidly; and that when compressed by the shrinking alveolar area during exhalation, the film reduces surface tension to very low values. These observations suggest that surfactant vesicles adsorb quickly, and that during compression, the adsorbed film resists the tendency to collapse from the interface to form a three-dimensional bulk phase....

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

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

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.

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.

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.

ADSORPTION OF SURFACTANT ON CLAYS

Science.gov (United States)

Surfactants used to enhance remediation of soils by soil washing are often lost in the process. Neither the amount nor the cause of this loss is known. It is assumed that clays present in the soil are responsible for the loss of the surfactant. In this papere, adsorption prope...

Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death

Science.gov (United States)

Zhu, Xiaohong; Caplan, Jeffrey; Mamillapalli, Padmavathi; Czymmek, Kirk; Dinesh-Kumar, Savithramma P

2010-01-01

Programmed cell death (PCD) initiated at the pathogen-infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca2+-ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N- and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptor-mediated PCD. Our results indicate that ER-Ca2+-ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response. PMID:20075858

Splash Dynamics of Falling Surfactant-Laden Droplets

Science.gov (United States)

Sulaiman, Nur; Buitrago, Lewis; Pereyra, Eduardo

2017-11-01

Splashing dynamics is a common issue in oil and gas separation technology. In this study, droplet impact of various surfactant concentrations onto solid and liquid surfaces is studied experimentally using a high-speed imaging analysis. Although this area has been widely studied in the past, there is still not a good understanding of the role of surfactant over droplet impact and characterization of resulting splash dynamics. The experiments are conducted using tap water laden with anionic surfactant. The effects of system parameters on a single droplet impingement such as surfactant concentration (no surfactant, below, at and above critical micelle concentration), parent drop diameter (2-5mm), impact velocity and type of impact surface (thin and deep pool) are investigated. Image analysis technique is shown to be an effective technique for identification of coalescence to splashing transition. In addition, daughter droplets size distributions are analyzed qualitatively in the events of splashing. As expected, it is observed that the formation of secondary droplets is affected by the surfactant concentration. A summary of findings will be discussed.

Recovering hydrocarbons with surfactants from lignin

Energy Technology Data Exchange (ETDEWEB)

Naae, D.G.; Whittington, L.E.; Ledoux, W.A.; Debons, F.E.

1988-11-29

This patent describes a method of recovering hydrocarbons from an underground hydrocarbon formation penetrated by at least one injection well and at least one production well, which comprises: injecting into the formation through an injection well a surfactant slug comprising about 0.1% to about 10% by weight of surfactants produced from lignin, the surfactants produced by placing lignin in contact with water, converting the lignin into low molecular weight lignin phenols by reducing the lignin in the presence of a reducing agent of carbon monoxide or hydrogen creating a reduction reaction mixture comprising oil soluble lignin phenols, the reduction occurring at a temperature greater than about 200/sup 0/C and a pressure greater than about 100 psi, recovering the oil soluble lignin phenols from the reduction mixture, and converting the lignin phenols into lignin surfactants by a reaction selected from the group consisting of alkoxylation, sulfonation, sulfation, aklylation, sulfomethylation, and alkoxysulfation; injecting into the formation through the injection well a drive fluid to push the surfactant slug towards a production well; and recovering hydrocarbons at the production well.

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.

Undoped Polyaniline/Surfactant Complex for the Corrosion Prevention

Science.gov (United States)

Liu, Lo-Min; Levon, Kalle

1998-01-01

Due to the strict regulations on the usage of heavy metals as the additives in the coating industries, the search for effective organic corrosion inhibitors in replace of those metal additives has become essential. Electrically conducting polymers have been shown to be effective for corrosion prevention but the poor solubility of these intractable polymers has been a problem. We have explored a polyaniline/4-dodecylphenol complex (PANi/DDPh) to improve the dissolution and it has been shown to be an effective organic corrosion inhibitor. With the surfactant, DDPh, PANi could be diluted into the coatings and the properties of the coatings were affected. Emeraldine base (EB) form of PANi was also found to be oxidized by the hardener. The oxidized form of polyaniline provides improved corrosion protection of metals than that of emeraldine base since the value of the standard electrode potential for the oxidized form of PANi is higher than that of EB. Additionally, the surfactant improves the wet adhesion property between the coating and the metal surface.

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.

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

Arabidopsis ACA7, encoding a putative auto-regulated Ca(2+)-ATPase, is required for normal pollen development.

Science.gov (United States)

Lucca, Noel; León, Gabriel

2012-04-01

Microgametogenesis is a complex process that involves numerous well-coordinated cell activities, ending with the production of pollen grains. Pollen development has been studied at the cytological level in Arabidopsis and other plant species, where its temporal time course has been defined. However, the molecular mechanism underlying this process is still unclear, since a relative small number of genes and/or processes have been identified as essential for pollen development. We have designed a methodology to select candidate genes for functional analysis, based on transcriptomic data obtained from different stages of pollen development. From our analyses, we selected At2g22950 as a candidate gene; this gene encodes a protein belonging to the auto-regulated Ca(2+)-ATPase family, ACA7. Microarray data indicate that ACA7 is expressed exclusively in developing pollen grains, with the highest level of mRNA at the time of the second pollen mitosis. Our RT-PCR experiments showed that ACA7 mRNA is detected exclusively in developing flowers. Confocal microscopy experiments showed a plasma membrane localization for the recombinant GFP:ACA7 protein. We identified two different insertional mutant lines, aca7-1 and aca7-2; plants from both mutant lines displayed a normal vegetative development but showed large amounts of dead pollen grains in mature flowers assayed by Alexander's staining. Histological analysis indicated that abnormalities are detected after the first pollen mitosis and we found a strong correlation between ACA7 mRNA accumulation and the severity of the phenotype. Our results indicate that ACA7 is a plasma membrane protein that has an important role during pollen development, possibly through regulation of Ca(2+) homeostasis. © Springer-Verlag 2011

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.

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.

Modulation by K+ Plus NH4+ of microsomal (Na+, K+)-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

Science.gov (United States)

Leone, Francisco A; Bezerra, Thais M S; Garçon, Daniela P; Lucena, Malson N; Pinto, Marcelo R; Fontes, Carlos F L; McNamara, John C

2014-01-01

We investigate the synergistic stimulation by K(+) plus NH4 (+) of (Na(+), K(+))-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+), K(+))-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+) and NH4 (+) binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+), K(+))-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+) and NH4 (+) of gill (Na(+), K(+))-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+) during ontogenetic development in M. amazonicum.

Modulation by K+ Plus NH4+ of microsomal (Na+, K+-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae.

Directory of Open Access Journals (Sweden)

Francisco A Leone

Full Text Available We investigate the synergistic stimulation by K(+ plus NH4 (+ of (Na(+, K(+-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+, K(+-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+ and NH4 (+ binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+, K(+-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+, K(+-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+ and NH4 (+ of gill (Na(+, K(+-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+ during ontogenetic development in M. amazonicum.

Quantitative determination of Na sup + -K sup + -ATPase and other sarcolemmal components in muscle cells

Energy Technology Data Exchange (ETDEWEB)

Hansen, O.; Clausen, T. (Aarhus Univ. (Denmark))

1988-01-01

A recurring problem in the characterization of plasma membrane enzymes in tissues and cells is whether the samples tested are representative for the entire population of enzyme molecules present in the starting material. Measurements of ({sup 3}H)-ouabain binding, enzyme activity, and maximum transport capacity all indicate that the concentration of Na{sup +}-K{sup +} pumps in mammalian skeletal muscle is high. Studies on Na{sup +}-K{sup +}-ATPase activity in isolated sarcolemma, however, generally give little or no information on total cellular enzyme concentration. Due to the low and variable enzyme recovery, such subcellular preparations may, therefore, give misleading data on factors regulating Na{sup +}-K{sup +}-ATPase in heart and skeletal muscle cells. As the same isolation and purification procedures are used for the study of other sarcolemmal components, this inadequate recovery has general implications for statements on regulatory changes in the sarcolemmal composition of muscle cells. On the other hand, complete quantification of Na{sup +}-K{sup +}-ATPase in muscle tissue can now be achieved using simple procedures and the entire material. Recent studies have shown that regulatory changes in the entire population of Na{sup +}-K{sup +} pumps in muscle can be quantified in measurements of ({sup 3}H)-ouabain binding, K{sup +}-activated 3-O-methylfluorescein phosphatase activity, as well as maximum ouabain suppressible Na{sup +}-K{sup +} transport capacity.

Influence of metacide - surfactant complexes on agricultural crops

Directory of Open Access Journals (Sweden)

Orynkul Esimova

2014-12-01

Full Text Available The complexes based on surfactants and polyhexamethyleneguanidine hydrochloride (metacide are important for agriculture. This paper considers compositions of known bactericidal metacide with different surfactants: anionic surfactant sodium dodecylsulphate (DDSNa and nonionic surfactant Tween 80 (monooleate of oxyethylenated anhydrosorbitols. The effect of individual components and associates of metacide and surfactants on productivity and infection of cereals was studied. According to the study, the highest productivity and infection rate were shown by the associate of metacide and Tween-80. At concentration of Tween-80 in aqueous solution equal to 0.001% in combination with metacide, efficiency was 98% at 0% infection. The surface tension and the wetting of metacide, DDSNa, Tween-80, and associates of metacide with surfactants were studied. In comparison with individual components, metacide-DDSNa and metacide-Tween-80 associates have higher surface activity.

Physicochemical characteristics of PFC surfactants for dry decontamination

Energy Technology Data Exchange (ETDEWEB)

Oh, Won Jin; Lee, Chi Woo [Korea University, Seoul (Korea)

2001-04-01

Even the trace amount of the used nuclear fuels of high radioactivity are hazardous to the earth and humans. Perfluorocarbons and perfluorocarbon surfactants are emerging to be efficient chemicals in the dry decontamination process of the used fuels of high radioactivity. The theme was undertaken to increase the knowledge on perfluorocarbon surfactants to develop the perfluorocarbon system in the dry decontamination process in Korea. Several cationic and anionic pfc surfactants were synthesized. Effects of pfc surfactants on electrochemical etching of silicon were investigated to form porous silicons. Forces were measured between silicon surfaces and AFM tip in the absence and presence of pfc surfactants. 7 refs., 10 figs. (Author)

Tunable, antibacterial activity of silicone polyether surfactants.

Science.gov (United States)

Khan, Madiha F; Zepeda-Velazquez, Laura; Brook, Michael A

2015-08-01

Silicone surfactants are used in a variety of applications, however, limited data is available on the relationship between surfactant structure and biological activity. A series of seven nonionic, silicone polyether surfactants with known structures was tested for in vitro antibacterial activity against Escherichia coli BL21. The compounds varied in their hydrophobic head, comprised of branched silicone structures with 3-10 siloxane linkages and, in two cases, phenyl substitution, and hydrophilic tail of 8-44 poly(ethylene glycol) units. The surfactants were tested at three concentrations: below, at, and above their Critical Micelle Concentrations (CMC) against 5 concentrations of E. coli BL21 in a three-step assay comprised of a 14-24h turbidometric screen, a live-dead stain and viable colony counts. The bacterial concentration had little effect on antibacterial activity. For most of the surfactants, antibacterial activity was higher at concentrations above the CMC. Surfactants with smaller silicone head groups had as much as 4 times the bioactivity of surfactants with larger groups, with the smallest hydrophobe exhibiting potency equivalent to sodium dodecyl sulfate (SDS). Smaller PEG chains were similarly associated with higher potency. These data link lower micelle stability and enhanced permeability of smaller silicone head groups to antibacterial activity. The results demonstrate that simple manipulation of nonionic silicone polyether structure leads to significant changes in antibacterial activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L.

Science.gov (United States)

Chen, Dan; Deng, Yingtian; Zhao, Jie

2012-01-15

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.