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Sample records for enzyme atpases promote

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-07-01

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  3. ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

    Science.gov (United States)

    Sehgal, Pankaj; Olesen, Claus; Møller, Jesper V

    2016-01-01

    Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

  4. Na+,K+-ATPase as the Target Enzyme for Organic and Inorganic Compounds

    Directory of Open Access Journals (Sweden)

    Tatjana Momić

    2008-12-01

    Full Text Available This paper gives an overview of the literature data concerning specific and non specific inhibitors of Na+,K+-ATPase receptor. The immobilization approaches developed to improve the rather low time and temperature stability of Na+,K+-ATPase, as well to preserve the enzyme properties were overviewed. The functional immobilization of Na+,K+-ATPase receptor as the target, with preservation of the full functional protein activity and access of various substances to an optimum number of binding sites under controlled conditions in the combination with high sensitive technology for the detection of enzyme activity is the basis for application of this enzyme in medical, pharmaceutical and environmental research.

  5. 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

  6. 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

  7. Subunit topology in the V type ATPase and related enzymes

    NARCIS (Netherlands)

    Chaban, Yuriy

    2005-01-01

    During the last decades impressive progress has been made in understanding of the catalytic mechanism of F-type ATP synthase, which is the key enzyme in the energy metabolism of eukaryotes and most bacteria. This enzyme catalyzes the final step in the process of oxidative phosphorylation in bacteria

  8. 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.

  9. Methods for Creating and Animating a Computer Model Depicting the Structure and Function of the Sarcoplasmic Reticulum Calcium ATPase Enzyme.

    Science.gov (United States)

    Chen, Alice Y.; McKee, Nancy

    1999-01-01

    Describes the developmental process used to visualize the calcium ATPase enzyme of the sarcoplasmic reticulum which involves evaluating scientific information, consulting scientists, model making, storyboarding, and creating and editing in a computer medium. (Author/CCM)

  10. Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

    Science.gov (United States)

    Metz, D C; Pradhan, T K; Mrozinski, J E; Jensen, R T; Turner, R J; Patto, R J; Gardner, J D

    1994-01-13

    We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

  11. Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

    Science.gov (United States)

    Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Adzhubei, Alexei A.; Burnysheva, Ksenia M.; Lakunina, Valentina A.; Kamanina, Yulia V.; Dergousova, Elena A.; Lopina, Olga D.; Ogunshola, Omolara O.; Bogdanova, Anna Yu.; Makarov, Alexander A.

    2016-06-01

    By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

  12. Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

    Science.gov (United States)

    Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

    2015-04-01

    Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  14. 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

  15. 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

  16. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors.

    Science.gov (United States)

    Chen, Ronald J Y; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason T C

    2011-02-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

  17. 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

  18. Phenylethynyl-butyltellurium inhibits the sulfhydryl enzyme Na+, K+ -ATPase: an effect dependent on the tellurium atom.

    Science.gov (United States)

    Quines, Caroline B; Rosa, Suzan G; Neto, José S S; Zeni, Gilson; Nogueira, Cristina W

    2013-11-01

    Organotellurium compounds are known for their toxicological effects. These effects may be associated with the chemical structure of these compounds and the oxidation state of the tellurium atom. In this context, 2-phenylethynyl-butyltellurium (PEBT) inhibits the activity of the sulfhydryl enzyme, δ-aminolevulinate dehydratase. The present study investigated on the importance of the tellurium atom in the PEBT ability to oxidize mono- and dithiols of low molecular weight and sulfhydryl enzymes in vitro. PEBT, at high micromolar concentrations, oxidized dithiothreitol (DTT) and inhibited cerebral Na(+), K(+)-ATPase activity, but did not alter the lactate dehydrogenase activity. The inhibition of cerebral Na(+), K(+)-ATPase activity was completely restored by DTT. By contrast, 2-phenylethynyl-butyl, a molecule without the tellurium atom, neither oxidized DTT nor altered the Na(+), K(+)-ATPase activity. In conclusion, the tellurium atom of PEBT is crucial for the catalytic oxidation of sulfhydryl groups from thiols of low molecular weight and from Na(+), K(+)-ATPase.

  19. Binding of ouabain and marinobufagenin leads to different structural changes in Na,K-ATPase and depends on the enzyme conformation.

    Science.gov (United States)

    Klimanova, Elizaveta A; Petrushanko, Irina Yu; Mitkevich, Vladimir A; Anashkina, Anastasia A; Orlov, Sergey N; Makarov, Alexander A; Lopina, Olga D

    2015-09-14

    Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types - marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Active compounds in Chinese herbs and medicinal animal products which promote blood circulation via inhibition of Na+, K+-ATPase.

    Science.gov (United States)

    Tzen, Jason Tc; Chen, Ronald Jy; Chung, Tse-Yu; Chen, Yi-Ching; Lin, Nan-Hei

    2010-01-01

    The therapeutic effect of cardiac glycosides for congestive heart failure lies in their reversible inhibition on Na+, K+-ATPase located in human myocardium. Several steroid-like compounds containing a core structure similar to cardiac glycosides have been found in many Chinese herbs and medicinal animal products conventionally used to promote blood circulation. They are putatively responsible for the therapeutic effect of those medicinal products via the same mechanism of inhibiting Na+, K+-ATPase. Inhibitory potency on Na+, K+-ATPase by ginsenosides, one of the identified steroid-like compounds, is significantly affected by sugar attachment that might cause steric hindrance of their binding to Na+, K+-ATPase. Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, substantially inhibit Na+, K+-ATPase. However, their inhibitory potency is abolished when sugar moieties are linked to the C-6 or C-20 position of the steroid-like structure. In contrast, no appreciable contents of steroid-like compounds are found in danshen, a well-known Chinese herb traditionally regarded as an effective medicine promoting blood circulation. Instead, magnesium lithospermate B (MLB), the major soluble ingredient in danshen, is assumed to be responsible for the therapeutic effect by inhibiting Na+, K+-ATPase in a manner comparable to cardiac glycosides. Neuroprotective effects of cardiac glycosides, ginsenosides and MLB against ischemic stroke were accordingly observed in a cortical brain slice-based assay model. Whether the neuroprotection is also triggered by inhibition of Na+, K+-ATPase remains to be investigated. Molecular modeling suggests that cardiac glycosides, ginsenosides and MLB presumably bind to the same extracellular pocket of the Na+, K+-ATPase alpha subunit.

  1. Effects of alkali metal cations on phospho-enzyme levels and [3H] ouabain binding to (Na+ + K+)-ATPase.

    Science.gov (United States)

    Han, C S; Tobin, T; Akera, T; Brody, T M

    1976-05-13

    The effects of several alkali metal cations on the relationship between steady state phospho-enzyme levels and initial velocity and equilibrium levels of [3H]-ouabain binding to (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3.) were examined. Only Na+ increased both phospho-enzyme and [3H] ouabain binding levels above those observed in the presence of Mg2+ alone. While Na+ stimulated phosphorylation with an apparent Km of about 1 mM, its stimulation of [3H] ouabain binding was biphasic, the lower Km for stimulation corresponding to the Km for formation of phospho-enzyme. Among the other alkali metal cations, potassium, rubidium and lithium were at least eight times more effect in reducing phospho-enzyme levels than in reducing [3H] ouabain binding. This discrepancy is not due to the stability of the enzyme-ouabain complex, nor to any action on the rates of formation or dissociation of the enzyme-ouabain complex. The data thus suggest that [3H] ouabain interacts with the K+, Rb+ or Li+ -enzyme complexes. For Li+, this hypothesis is further supported by the observation that Li+ can cirectly increase the equilibrium level of [3H] ouabain binding to this enzyme under certain conditions.

  2. 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

  3. 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

  4. Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+ -ATPase.

    Science.gov (United States)

    Chen, Ronald J Y; Chung, Tse-yu; Li, Feng-yin; Yang, Wei-hung; Jinn, Tzyy-rong; Tzen, Jason T C

    2010-06-01

    To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. The inhibitory potency of ouabain and the identified steroid-like compounds on Na(+)/K(+)-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na(+)/K(+)-ATPase. All the examined steroid-like compounds displayed more or less inhibition on Na(+)/K(+)-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na(+)/K(+)-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K(+) binding sites of Na(+)/K(+)-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na(+)/K(+)-ATPase. Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na(+)/K(+)-ATPase.

  5. Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress.

    Science.gov (United States)

    Singh, Raja B; Hryshko, Larry; Freed, Darren; Dhalla, Naranjan S

    2012-02-01

    We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.

  6. 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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  8. Cysteine residues 244 and 458–459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

    Directory of Open Access Journals (Sweden)

    Irina Yu. Petrushanko

    2017-10-01

    Full Text Available Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD and nucleotide binding domain (NBD of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG. Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459 were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor

  9. 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

  10. Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis

    Directory of Open Access Journals (Sweden)

    Vladimir A. Mitkevich

    2016-01-01

    Full Text Available Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

  11. Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis.

    Science.gov (United States)

    Mitkevich, Vladimir A; Petrushanko, Irina Yu; Poluektov, Yuri M; Burnysheva, Ksenia M; Lakunina, Valentina A; Anashkina, Anastasia A; Makarov, Alexander A

    2016-01-01

    Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

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

    Science.gov (United States)

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

    2014-01-07

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

  13. The two faces of endogenous DNA editing enzymes: Promoting ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    The two faces of endogenous DNA editing enzymes: Promoting gene mutations as well as genome repair. Type B lymphocytes are a specific type of white blood cell within our immune system. They produce and export antibodies which seek out, attach to, and neutralize microbes and toxins. A unique way that B ...

  14. Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration.

    Science.gov (United States)

    Zhai, Binyuan; DuPrez, Kevin; Doukov, Tzanko I; Li, Huan; Huang, Mengting; Shang, Guijun; Ni, Jinfeng; Gu, Lichuan; Shen, Yulong; Fan, Li

    2017-04-07

    Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    Science.gov (United States)

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. 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.

  17. Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

    Science.gov (United States)

    Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

    1999-07-01

    The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

  18. Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells.

    Science.gov (United States)

    Dang, Donna; Prasad, Hari; Rao, Rajini

    2017-11-01

    Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca 2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca 2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer. © 2017 Wiley Periodicals, Inc.

  19. Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region.

    Science.gov (United States)

    Morsomme, P; Dambly, S; Maudoux, O; Boutry, M

    1998-12-25

    The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

  20. 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.

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

    Science.gov (United States)

    Majumdar, Arkajo; Kar, Rup Kumar

    2018-07-01

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

  2. DUOX enzyme activity promotes AKT signalling in prostate cancer cells.

    Science.gov (United States)

    Pettigrew, Christopher A; Clerkin, John S; Cotter, Thomas G

    2012-12-01

    Reactive oxygen species (ROS) and oxidative stress are related to tumour progression, and high levels of ROS have been observed in prostate tumours compared to normal prostate. ROS can positively influence AKT signalling and thereby promote cell survival. The aim of this project was to establish whether the ROS generated in prostate cancer cells positively regulate AKT signalling and enable resistance to apoptotic stimuli. In PC3 cells, dual oxidase (DUOX) enzymes actively generate ROS, which inactivate phosphatases, thereby maintaining AKT phosphorylation. Inhibition of DUOX by diphenylene iodium (DPI), intracellular calcium chelation and small-interfering RNA (siRNA) resulted in lower ROS levels, lower AKT and glycogen synthase kinase 3β (GSK3β) phosphorylation, as well as reduced cell viability and increased susceptibility to apoptosis stimulating fragment (FAS) induced apoptosis. This report shows that ROS levels in PC3 cells are constitutively maintained by DUOX enzymes, and these ROS positively regulate AKT signalling through inactivating phosphatases, leading to increased resistance to apoptosis.

  3. The yeast H+-ATPase Pma1 promotes Rag/Gtr-dependent TORC1 activation in response to H+-coupled nutrient uptake.

    Science.gov (United States)

    Saliba, Elie; Evangelinos, Minoas; Gournas, Christos; Corrillon, Florent; Georis, Isabelle; André, Bruno

    2018-03-23

    The yeast Target of Rapamycin Complex 1 (TORC1) plays a central role in controlling growth. How amino acids and other nutrients stimulate its activity via the Rag/Gtr GTPases remains poorly understood. We here report that the signal triggering Rag/Gtr-dependent TORC1 activation upon amino-acid uptake is the coupled H + influx catalyzed by amino-acid/H + symporters. H + -dependent uptake of other nutrients, ionophore-mediated H + diffusion, and inhibition of the vacuolar V-ATPase also activate TORC1. As the increase in cytosolic H + elicited by these processes stimulates the compensating H + -export activity of the plasma membrane H + -ATPase (Pma1), we have examined whether this major ATP-consuming enzyme might be involved in TORC1 control. We find that when the endogenous Pma1 is replaced with a plant H + -ATPase, H + influx or increase fails to activate TORC1. Our results show that H + influx coupled to nutrient uptake stimulates TORC1 activity and that Pma1 is a key actor in this mechanism. © 2018, Saliba et al.

  4. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase

    DEFF Research Database (Denmark)

    Thastrup, Ole; Cullen, P J; Drøbak, B K

    1990-01-01

    . This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum...

  5. p97 Composition Changes Caused by Allosteric Inhibition Are Suppressed by an On-Target Mechanism that Increases the Enzyme's ATPase Activity.

    Science.gov (United States)

    Her, Nam-Gu; Toth, Julia I; Ma, Chen-Ting; Wei, Yang; Motamedchaboki, Khatereh; Sergienko, Eduard; Petroski, Matthew D

    2016-04-21

    The AAA ATPase p97/VCP regulates protein homeostasis using a diverse repertoire of cofactors to fulfill its biological functions. Here we use the allosteric p97 inhibitor NMS-873 to analyze its effects on enzyme composition and the ability of cells to adapt to its cytotoxicity. We found that p97 inhibition changes steady state cofactor-p97 composition, leading to the enrichment of a subset of its cofactors and polyubiquitin bound to p97. We isolated cells specifically insensitive to NMS-873 and identified a new mutation (A530T) in p97. A530T is sufficient to overcome the cytotoxicity of NMS-873 and alleviates p97 composition changes caused by the molecule but not other p97 inhibitors. This mutation does not affect NMS-873 binding but increases p97 catalytic efficiency through altered ATP and ADP binding. Collectively, these findings identify cofactor-p97 interactions sensitive to p97 inhibition and reveal a new on-target mechanism to suppress the cytotoxicity of NMS-873. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation).

    Science.gov (United States)

    Vera-Estrella, R.; Barkla, B. J.; Higgins, V. J.; Blumwald, E.

    1994-01-01

    Elicitor preparations containing the avr5 gene products from race 4 of Cladosporium fulvum and tomato (Lycopersicon esculentum L.) cells near isogenic for the resistance gene Cf5 were used to investigate events following the treatment of host plasma membranes with elicitor. A 4-fold increase in H+-ATPase activity, coincident with the acidification of the extracellular medium, was detected immediately after elicitor treatment. The elicitor-induced stimulation of the plasma membrane H+-ATPase was inhibited by okadaic acid but not by staurosporine, suggesting that protein dephosphorylation was required for increased H+-ATPase activity. This observation was confirmed by [gamma]-32P labeling and immunodetection of the plasma membrane H+-ATPase. Effects of guanidine nucleotide analogs and mastoparan on the ATPase activity suggested the role of GTP-binding proteins in mediating the putative elicitor-receptor binding, resulting in activation of a phosphatase(s), which in turn stimulates the plasma membrane H+-ATPase by dephosphorylation. PMID:12232073

  7. Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

    DEFF Research Database (Denmark)

    Henriksen, Carina; Kjaer-Sorensen, Kasper; Einholm, Anja Pernille

    2013-01-01

    Na+/K+-ATPase maintains electrochemical gradients of Na+ and K+ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model...... of the arginine with the C-terminus, stabilizing one of the Na+ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue....... Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which...

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

    Science.gov (United States)

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

    2011-07-01

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

  9. Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

    Directory of Open Access Journals (Sweden)

    Carina Henriksen

    Full Text Available Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4 encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by

  10. Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H(+)-ATPase in activated and basal-level enzyme preparations

    Czech Academy of Sciences Publication Activity Database

    Lapathitis, Georgios; Tanfani, F.; Kotyk, Arnošt; Bertoli, E.

    2001-01-01

    Roč. 505, č. 1 (2001), s. 155-158 ISSN 0014-5793 R&D Projects: GA ČR GA204/98/0474 Keywords : H+-ATPase * plasma membrane * two-dimensional gel electrophoresis Subject RIV: CE - Biochemistry Impact factor: 3.644, year: 2001

  11. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  12. 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.

  13. 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

  14. Promoting effects of phenobarbital on the enzyme-altered foci induced by intrahepatic γ-ray-irradiation in the rat liver

    International Nuclear Information System (INIS)

    Ida, Katsuya; Nakamura, Satoshi; Muro, Hiroyuki; Takai, Michikatsu; Kaneko, Masao

    1995-01-01

    Radiation-induced carcinogenesis of the rat liver using iridium-192 seeds as an intrahepatic radioactive source was studied by enzyme histochemical means. Rats were divided into six groups according to various combinations of one or two iridium-192 or stainless steel seeds and whether they were given a diet containing 0.05% phenobarbital (PB) or a basal diet (BD). Each group were sacrificed at 20, 40, and 60 weeks after intrahepatic insertion of the iridium-192 or stainless steel seeds. γ-Glutamyl transpeptidase (GGT), glucose-6-phosphatase (G6Pase), and adenosine triphosphatase (ATPase) were stained in the liver tissues, and GGT-positive foci were quantified. Liver neoplasm was not evident, but enzyme-altered foci (EAF) were induced by γ-ray irradiation. At every point (20, 40, and 60 weeks) after the insertion of the seeds, the GGT-positive area was larger in the rats given PB than those given BD. Moreover, despite the iridium-192 radioactivity decay, EAF developed continuously in the rats given PB, and persisted in those given BD from 40 to 60 weeks after insertion. These results indicated that phenobarbital promotes the development of EAF initiated by irradiation, as it promotes the process of chemical carcinogenesis in the rat liver. (author)

  15. Structure-function relationships in the Na,K-ATPase α subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    International Nuclear Information System (INIS)

    Price, E.M.; Lingrel, J.B.

    1988-01-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the α1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat α1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase α subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep α1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep α1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep α1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat α1 cDNA, the rat/sheep chimera, or the mutant sheep α1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86 Rb + uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase α subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep α1 subunit (glutamine and asparagine) are somehow involved in ouabain binding

  16. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  17. Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation

    Science.gov (United States)

    Shanks, Julia; Herring, Neil; Johnson, Errin; Liu, Kun; Li, Dan

    2017-01-01

    Reduced cardiomyocyte excitation–contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague–Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague–Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; Pneurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; Pneurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined. PMID:28223472

  18. 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.

  19. 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.

  20. Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes

    Directory of Open Access Journals (Sweden)

    Sheikh Hasna Habib

    2016-01-01

    Full Text Available Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-promoting rhizobacteria (PGPR containing 1-aminocyclopropane-1-carboxylate (ACC deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress.

  1. 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.

  2. 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.

  3. 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

  4. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

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

  5. The endogenous proteoglycan-degrading enzyme ADAMTS-4 promotes functional recovery after spinal cord injury

    Directory of Open Access Journals (Sweden)

    Tauchi Ryoji

    2012-03-01

    Full Text Available Abstract Background Chondroitin sulfate proteoglycans are major inhibitory molecules for neural plasticity under both physiological and pathological conditions. The chondroitin sulfate degrading enzyme chondroitinase ABC promotes functional recovery after spinal cord injury, and restores experience-dependent plasticity, such as ocular dominance plasticity and fear erasure plasticity, in adult rodents. These data suggest that the sugar chain in a proteoglycan moiety is essential for the inhibitory activity of proteoglycans. However, the significance of the core protein has not been studied extensively. Furthermore, considering that chondroitinase ABC is derived from bacteria, a mammalian endogenous enzyme which can inactivate the proteoglycans' activity is desirable for clinical use. Methods The degradation activity of ADAMTS-4 was estimated for the core proteins of chondroitin sulfate proteoglycans, that is, brevican, neurocan and phosphacan. To evaluate the biological significance of ADMATS-4 activity, an in vitro neurite growth assay and an in vivo neuronal injury model, spinal cord contusion injury, were employed. Results ADAMTS-4 digested proteoglycans, and reversed their inhibition of neurite outgrowth. Local administration of ADAMTS-4 significantly promoted motor function recovery after spinal cord injury. Supporting these findings, the ADAMTS-4-treated spinal cord exhibited enhanced axonal regeneration/sprouting after spinal cord injury. Conclusions Our data suggest that the core protein in a proteoglycan moiety is also important for the inhibition of neural plasticity, and provides a potentially safer tool for the treatment of neuronal injuries.

  6. Quaternary Benzyltriethylammonium Ion Binding to the Na,K-ATPase: a Tool to Investigate Extracellular K+ Binding Reactions†

    Science.gov (United States)

    Peluffo, R. Daniel; González-Lebrero, Rodolfo M.; Kaufman, Sergio B.; Kortagere, Sandhya; Orban, Branly; Rossi, Rolando C.; Berlin, Joshua R.

    2009-01-01

    This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (VM)-dependent inhibition and tested the prediction that such a VM-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K+ activation of Na,K-ATPase activity and steady-state 86Rb+ occlusion. The initial rate of 86Rb+ occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb+, a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a VM-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K+-K+ exchange by the Na,K-ATPase, step changes in VM elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K+ congener, Tl+. pNBTEA- and Tl+-dependent transient currents both displayed saturation of charge moved at extreme negative and positive VM, equivalence of charge moved during and after step changes in VM, and similar apparent valence. The rate constant (ktot) for Tl+-dependent transient current asymptotically approached a minimum value at positive VM. In contrast, ktot for pNBTEA-dependent transient current was a “U”-shaped function of VM with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly-accessible sites, one of them located at K+ binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme. PMID:19621894

  7. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    Science.gov (United States)

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

  8. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

    Directory of Open Access Journals (Sweden)

    Geisler Markus

    1998-01-01

    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  9. 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....

  10. 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.)

  11. Changes of initiation, promotion and metastatic enzyme system in human breast cancer with the proton irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Sohn, Y. H.; Kim, S. W.; Lee, K. S.; Mo, J. Y. [Dongguk University, Seoul (Korea, Republic of)

    2010-04-15

    Proton irradiations in the cells were significantly decreased cell viability but increased the QR activity in a dose-dependent manner. Cell viability was 92.3%, 88.4%, 81.8%, 72.4%, 68.9% at doses of 0.5, 2, 8, 16, and 32 Gy, respectively. At doses of 2, 8, 16, and 32 Gy, QR activity was increased 1.27-, 1.31-, 1.45- and 2.08-fold. However, negligible GST activity in the cells was detected and the activity was not changed by proton irradiation. Proton irradiation also increased GSH contents by 1.18- and 1.21-fold at doses of 0.5 and 2 Gy. In contrast, the ODC activity, a key enzyme in polyamine biosynthesis and tumor promotion, was decreased in a dose-dependent manner. We also investigated anti-metastatic effects of proton beam irradiation in breast cancer cells. Invasion and wound healing assay showed that metastatic activities in breast cancer cells were significantly decreased in a dose-dependent manner by proton beam irradiation. In zymography of MMP-9, the activity was slightly diminished. These results suggest that breast cancer chemopreventive potential was increased with proton irradiation by increasing the QR activity and the GSH levels and by inhibiting the ODC activity.

  12. Changes of initiation, promotion and metastatic enzyme system in human breast cancer with the proton irradiation

    International Nuclear Information System (INIS)

    Sohn, Y. H.; Kim, S. W.; Lee, K. S.; Mo, J. Y.

    2010-04-01

    Proton irradiations in the cells were significantly decreased cell viability but increased the QR activity in a dose-dependent manner. Cell viability was 92.3%, 88.4%, 81.8%, 72.4%, 68.9% at doses of 0.5, 2, 8, 16, and 32 Gy, respectively. At doses of 2, 8, 16, and 32 Gy, QR activity was increased 1.27-, 1.31-, 1.45- and 2.08-fold. However, negligible GST activity in the cells was detected and the activity was not changed by proton irradiation. Proton irradiation also increased GSH contents by 1.18- and 1.21-fold at doses of 0.5 and 2 Gy. In contrast, the ODC activity, a key enzyme in polyamine biosynthesis and tumor promotion, was decreased in a dose-dependent manner. We also investigated anti-metastatic effects of proton beam irradiation in breast cancer cells. Invasion and wound healing assay showed that metastatic activities in breast cancer cells were significantly decreased in a dose-dependent manner by proton beam irradiation. In zymography of MMP-9, the activity was slightly diminished. These results suggest that breast cancer chemopreventive potential was increased with proton irradiation by increasing the QR activity and the GSH levels and by inhibiting the ODC activity.

  13. 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.

  14. 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/.

  15. Na,K-ATPase reconstituted in ternary liposome: the presence of cholesterol affects protein activity and thermal stability.

    Science.gov (United States)

    Yoneda, Juliana Sakamoto; Rigos, Carolina Fortes; de Lourenço, Thaís Fernanda Aranda; Sebinelli, Heitor Gobbi; Ciancaglini, Pietro

    2014-12-15

    Differential scanning calorimetry (DSC) was applied to investigate the effect of cholesterol on the thermotropic properties of the lipid membrane (DPPC and DPPE). The thermostability and unfolding of solubilized and reconstituted Na,K-ATPase in DPPC:DPPE:cholesterol-liposomes was also studied to gain insight into the role of cholesterol in the Na,K-ATPase modulation of enzyme function and activity. The tertiary system (DPPC:DPPE:cholesterol) (molar ratio DPPC:DPPE equal 1:1) when cholesterol content was increased from 0% up to 40% results in a slight decrease in the temperature of transition and enthalpy, and an increase in width. We observed that, without heating treatment, at 37°C, the activity was higher for 20mol% cholesterol. However, thermal inactivation experiments showed that the enzyme activity loss time depends on the cholesterol membrane content. The unfolding of the enzyme incorporated to liposomes of DPPC:DPPE (1:1mol) with different cholesterol contents, ranging from 0% to 40% mol was also studied by DSC. Some differences between the thermograms indicate that the presence of lipids promotes a conformational change in protein structure and this change is enough to change the way Na,K-ATPase thermally unfolds. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. 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.

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

    Science.gov (United States)

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

    2016-11-01

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

  18. 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

  19. Potential of plant growth promoting rhizobacteria and chemical fertilizers on soil enzymes and plant growth

    International Nuclear Information System (INIS)

    Nosheen, A.; Bano, A.

    2014-01-01

    The present investigation deals with the role of Plant Growth Promoting Rhizobacteria and chemical fertilizers alone or in combination on urease, invertase and phosphatase activities of rhizospheric soil and also on general impact on growth of safflower cvv. Thori and Saif-32. The PGPR (Azospirillum brasilense and Azotobacter vinelandii) were applied at 10/sup 6/ cells/mL as seed inoculation prior to sowing. Chemical fertilizers were applied at full (Urea 60 Kg ha/sup -1/ and Diammonium phosphate (DAP) 30 Kg ha/sup -1/), half (Urea 30 Kg ha/sup -1/ and DAP 15 Kg ha/sup -1/) and quarter doses (Urea 15 Kg ha-1 and DAP 7.5 Kg ha/sup -1/) during sowing. The chemical fertilizers and PGPR enhanced urease and invertase activities of soil. Presence of PGPR in combination with quarter and half doses of chemical fertilizers further augmented their effect on soil enzymes activities. The soil phosphatase activity was greater in Azospirillum and Azotobacter in combination with half dose of chemical fertilizers. Maximum increase in leaf melondialdehyde content was recorded in full dose of chemical fertilizers whereas coinoculation treatment exhibited significant reduction in cv. Thori. Half and quarter dose of chemical fertilizers increased the shoot length of safflower whereas maximum increase in leaf protein was recorded in Azotobacter in combination with full dose of chemical fertilizers. Root length was improved by Azospirillum and Azotobacter in combination with quarter dose of chemical fertilizers. Leaf area and chlorophyll contents were significantly improved by Azotobacter in combination with half dose of chemical fertilizers. It is inferred that PGPR can supplement 50 % chemical fertilizers for better plant growth and soil health. (author)

  20. 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

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

    Science.gov (United States)

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

    2010-01-01

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

  2. Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance.

    Directory of Open Access Journals (Sweden)

    Alexander G Bulakhov

    Full Text Available Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO displaying a synergism with cellulases.Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL, and eglIV, encoding LPMO (formerly endoglucanase IV from Trichoderma reesei (TrLPMO, were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3 varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples. The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10-43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable

  3. 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.

  4. 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.

  5. 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.

  6. 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...

  7. In vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues

    International Nuclear Information System (INIS)

    Trottman, C.H.; Rao, K.S.P.; Morrow, W.; Uzodinma, J.E.; Desaiah, D.

    1985-01-01

    In vitro effects of toxaphene on Ca 2+ -ATPase activity and 45 Ca 2+ -uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca 2+ -ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca 2+ -ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca 2+ -ATPase activity non-competetively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial 45 Ca 2+ -uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca 2+ -ATPase and calcium transport in heart, kidney and liver tissues of rat. 19 references, 5 figures

  8. The effect of near-UV light on Na-K-ATPase of the rat lens

    International Nuclear Information System (INIS)

    Torriglia, A.; Zigman, S.

    1988-01-01

    The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a mono-exponential function, the existence of two forms of Na-K-ATPase in the lens is discussed. (author)

  9. Effect of near-UV light on Na-K-ATPase of the rat lens

    Energy Technology Data Exchange (ETDEWEB)

    Torriglia, A.; Zigman, S.

    1988-06-01

    The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a mono-exponential function, the existence of two forms of Na-K-ATPase in the lens is discussed.

  10. Evaluation of Na+, K+-ATPase activity in the brain of young rats after acute administration of fenproporex

    OpenAIRE

    Rezin, Gislaine T.; Scaini, Giselli; Gonçalves, Cinara L.; Ferreira, Gabriela K.; Cardoso, Mariane R.; Ferreira, Andréa G.K.; Cunha, Maira J.; Schmitz, Felipe; Varela, Roger B.; Quevedo, João; Wyse, Angela T.S.; Streck, Emilio L.

    2013-01-01

    Objectives: Fenproporex is an amphetamine-based anorectic which is rapidly converted into amphetamine in vivo. Na+, K+-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that the effects of fenproporex on brain metabolism are poorly known and that Na+, K+-ATPase is essential for normal brain function, this study sought to evaluate the effect of this drug on Na+, K+-ATPase activity in the hippocampus, hypothalamus, prefrontal cortex, and striatum of youn...

  11. Caffeine prevents high-intensity exercise-induced increase in enzymatic antioxidant and Na+-K+-ATPase activities and reduction of anxiolytic like-behaviour in rats.

    Science.gov (United States)

    Vieira, Juliano M; Carvalho, Fabiano B; Gutierres, Jessié M; Soares, Mayara S P; Oliveira, Pathise S; Rubin, Maribel A; Morsch, Vera M; Schetinger, Maria Rosa; Spanevello, Roselia M

    2017-11-01

    Here we investigated the impact of chronic high-intensity interval training (HIIT) and caffeine consumption on the activities of Na + -K + -ATPase and enzymes of the antioxidant system, as well as anxiolytic-like behaviour in the rat brain. Animals were divided into groups: control, caffeine (4 mg/kg), caffeine (8 mg/kg), HIIT, HIIT plus caffeine (4 mg/kg) and HIIT plus caffeine (8 mg/kg). Rats were trained three times per week for 6 weeks, and caffeine was administered 30 minutes before training. We assessed the anxiolytic-like behaviour, Na + -K + -ATPase, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) in the brain. HIIT-induced anxiolytic-like behaviour increased Na + -K + -ATPase and GPx activities and TBARS levels, altered the activities of SOD and CAT in different brain regions, and decreased GSH levels. Caffeine, however, elicited anxiogenic-like behaviour and blocked HIIT effects. The combination of caffeine and HIIT prevented the increase in SOD activity in the cerebral cortex and GPx activity in three brain regions. Our results show that caffeine promoted anxiogenic behaviour and prevented HIIT-induced changes in the antioxidant system and Na + -K + -ATPase activities.

  12. In Silico Docking of Small-Molecule Inhibitors to the Escherichia coli Type III Secretion System EscN ATPase

    Science.gov (United States)

    2014-07-01

    Adenosine triphosphatase (ATPase) Broad-spectrum antibiotic Drug discovery Enzyme inhibitors Enzyme structure Injectosome Molecular modeling Protein...Kagawa, Y.; Yoshida, M. The Crystal Structure of the Nucleotide- Free Alpha 3 Beta 3 Subcomplex of F1-ATPase from the Thermophilic Bacillus PS3 is...Kinases and other ATP- Requiring Enzymes and a Common Nucleotide Binding Fold. EMBO J. 1982, 1, 945–951. Zarivach, R.; Vuckovic, M.; Deng, W

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Enzyme-treated asparagus extract promotes expression of heat shock protein and exerts antistress effects.

    Science.gov (United States)

    Ito, Tomohiro; Maeda, Takahiro; Goto, Kazunori; Miura, Takehito; Wakame, Koji; Nishioka, Hiroshi; Sato, Atsuya

    2014-03-01

    A novel enzyme-treated asparagus extract (ETAS) has been developed as a functional material produced from asparagus stem. Studies were conducted to determine the effect of ETAS on heat shock protein 70 (HSP70) expression and alleviation of stress. HeLa cells were treated with ETAS, and HSP70 mRNA and protein levels were measured using a reverse transcription-polymerase chain reaction (RT-PCR) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. ETAS showed significant increases in HSP70 mRNA at more than 0.125 mg/mL and the protein at more than 1.0 mg/mL. The antistress effect was evaluated in a murine sleep-deprivation model. A sleep-deprivation stress load resulted in elevation of blood corticosterone and lipid peroxide concentrations, while supplementation with ETAS at 200 and 1000 mg/kg body weight was associated with significantly reduced levels of both stress markers, which were in the normal range. The HSP70 protein expression level in mice subjected to sleep-deprivation stress and supplemented with ETAS was significantly enhanced in stomach, liver, and kidney, compared to ETAS-untreated mice. A preliminary and small-sized human study was conducted among healthy volunteers consuming up to 150 mg/d of ETAS daily for 7 d. The mRNA expression of HSP70 in peripheral leukocytes was significantly elevated at intakes of 100 or 150 mg/d, compared to their baseline levels. Since HSP70 is known to be a stress-related protein and its induction leads to cytoprotection, the present results suggest that ETAS might exert antistress effects under stressful conditions, resulting from enhancement of HSP70 expression. © 2014 Institute of Food Technologists®

  19. The influence of blood plasma of irradiated animals on activity of Ca2+ - ATPase and Mg2+ - ATPase in plasma membrane of thymocytes

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1994-01-01

    Rats were irradiated at doses 1.5, 4.0, 7.0 and 10 Gy. After 1, 8, 15, 22 and 30 days the effect of blood plasma on activity of Ca 2+ -ATPase and Mg 2+ -ATPase in plasma membrane of thymocytes was investigated. It was found that the raise of irradiation dose leads to increasing of blood plasma effect on membrane-bound enzymes

  20. 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.

  1. 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

  2. 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.

  3. Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

    NARCIS (Netherlands)

    Dalla, S.; Swarts, H.G.P.; Koenderink, J.B.; Dobler, S.

    2013-01-01

    Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of

  4. Yeast Extract Promotes Cell Growth and Induces Production of Polyvinyl Alcohol-Degrading Enzymes

    Directory of Open Access Journals (Sweden)

    Min Li

    2011-01-01

    Full Text Available Polyvinyl alcohol-degrading enzymes (PVAases have a great potential in bio-desizing processes for its low environmental impact and low energy consumption. In this study, the effect of yeast extract on PVAases production was investigated. A strategy of four-point yeast extract addition was developed and applied to maximize cell growth and PVAases production. As a result, the maximum dry cell weight achieved was 1.48 g/L and the corresponding PVAases activity was 2.99 U/mL, which are 46.5% and 176.8% higher than the control, respectively. Applying this strategy in a 7 L fermentor increased PVAases activity to 3.41 U/mL. Three amino acids (glycine, serine, and tyrosine in yeast extract play a central role in the production of PVAases. These results suggest that the new strategy of four-point yeast extract addition could benefit PVAases production.

  5. Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Reinhard Klein

    2008-01-01

    Full Text Available Gene directed-enzyme prodrug therapy (GDEPT is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1 metabolizes the prodrugs cyclophosphamide (CPA and ifosfamide (IFA to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

  6. Functional analysis of a potential regulatory K+-binding site in the Na+, K+-ATPase

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Vilsen, Bente

    The Na+, K+-ATPase functions by actively transporting 3 Na+ ions out of and 2 K+ ions into the cell, thereby creating ion gradients crucial for many physiological processes. Recently, a combined structural and functional study of the closely related Ca2+-ATPase indicated the presence...... of a regulatory K+-binding site in the P-domain of the enzyme, identifying E732 as being of particular importance (Sorensen, Clausen et al. 2004). In addition, P709 is thought to play a significant role in the structural organization of this site. Both E732 and P709 are highly conserved among P-type ATPases (E732...... is present as either glutamic acid or aspartic acid), which supports their importance and additionally raises the question whether this site may play a general role among P-type ATPases. In Na+, K+-ATPase, K+ functions directly as a substrate for membrane binding sites, however, an additional regulatory...

  7. The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: Involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end

    DEFF Research Database (Denmark)

    Fuglsang, Anja T; Borch, Jonas; Bych, Katrine

    2003-01-01

    14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine...... of the Arabidopsis plasma membrane H+-ATPase isoform 2 (AHA2). Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3. Among these...

  8. 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...

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    2010-02-01

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  16. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    Science.gov (United States)

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. 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.

  18. 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

  19. 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

  20. Evaluation of Na+, K+-ATPase activity in the brain of young rats after acute administration of fenproporex.

    Science.gov (United States)

    Rezin, Gislaine T; Scaini, Giselli; Gonçalves, Cinara L; Ferreira, Gabriela K; Cardoso, Mariane R; Ferreira, Andréa G K; Cunha, Maira J; Schmitz, Felipe; Varela, Roger B; Quevedo, João; Wyse, Angela T S; Streck, Emilio L

    2014-01-01

    Fenproporex is an amphetamine-based anorectic which is rapidly converted into amphetamine in vivo. Na+, K+-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that the effects of fenproporex on brain metabolism are poorly known and that Na+, K+-ATPase is essential for normal brain function, this study sought to evaluate the effect of this drug on Na+, K+-ATPase activity in the hippocampus, hypothalamus, prefrontal cortex, and striatum of young rats. Young male Wistar rats received a single injection of fenproporex (6.25, 12.5, or 25 mg/kg intraperitoneally) or polysorbate 80 (control group). Two hours after the last injection, the rats were killed by decapitation and the brain was removed for evaluation of Na+, K+-ATPase activity. Fenproporex decreased Na+, K+-ATPase activity in the striatum of young rats at doses of 6.25, 12.5, and 25 mg/kg and increased enzyme activity in the hypothalamus at the same doses. Na+, K+-ATPase activity was not affected in the hippocampus or prefrontal cortex. Fenproporex administration decreased Na+, K+-ATPase activity in the striatum even in low doses. However, in the hypothalamus, Na+, K+-ATPase activity was increased. Changes in this enzyme might be the result of the effects of fenproporex on neuronal excitability.

  1. Evaluation of Na+, K+-ATPase activity in the brain of young rats after acute administration of fenproporex

    Directory of Open Access Journals (Sweden)

    Gislaine T. Rezin

    2014-05-01

    Full Text Available Objectives: Fenproporex is an amphetamine-based anorectic which is rapidly converted into amphetamine in vivo. Na+, K+-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that the effects of fenproporex on brain metabolism are poorly known and that Na+, K+-ATPase is essential for normal brain function, this study sought to evaluate the effect of this drug on Na+, K+-ATPase activity in the hippocampus, hypothalamus, prefrontal cortex, and striatum of young rats. Methods: Young male Wistar rats received a single injection of fenproporex (6.25, 12.5, or 25 mg/kg intraperitoneally or polysorbate 80 (control group. Two hours after the last injection, the rats were killed by decapitation and the brain was removed for evaluation of Na+, K+-ATPase activity. Results: Fenproporex decreased Na+, K+-ATPase activity in the striatum of young rats at doses of 6.25, 12.5, and 25 mg/kg and increased enzyme activity in the hypothalamus at the same doses. Na+, K+-ATPase activity was not affected in the hippocampus or prefrontal cortex. Conclusion: Fenproporex administration decreased Na+, K+-ATPase activity in the striatum even in low doses. However, in the hypothalamus, Na+, K+-ATPase activity was increased. Changes in this enzyme might be the result of the effects of fenproporex on neuronal excitability.

  2. 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/

  3. 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

  4. Biochemical characterization of the plasma membrane H+ - ATPase from red beet (Beta vulgaris) hypocotyl tissue

    International Nuclear Information System (INIS)

    Oleski, N.A.

    1986-01-01

    Several biochemical techniques including selective solubilization followed by gel filtration or various types of affinity chromatography, and antibody production were employed in an attempt to purify the plasma membrane H + - ATPase from red beet hypocotyl tissue. While the enzyme could not be purified using any of these methods, it was possible to successfully conduct a more detailed biochemical analysis of the H + - ATPase. The molecular weight and isoelectric point of the enzyme were determined using N,N'dicyclohexylcarbodiimide (DCCD) and a H + - ATPase antibody. When plasma membrane vesicles were incubated with 20 μM [ 14 C]-DCCD at 0 C, a single 97,000 dalton protein was apparent on a fluorograph. A close correlation between [ 14 C]-DCCD labelling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentrations 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 enzyme to be pH 6.5

  5. Melatonin Promotes Cheliped Regeneration, Digestive Enzyme Function, and Immunity Following Autotomy in the Chinese Mitten Crab, Eriocheir sinensis

    Directory of Open Access Journals (Sweden)

    Cong Zhang

    2018-03-01

    Full Text Available In the pond culture of juvenile Eriocheir sinensis, a high limb-impairment rate seriously affects the culture success. Therefore, it is particularly important to artificially promote limb regeneration. This study evaluated the effects of melatonin on cheliped regeneration, digestive ability, and immunity, as well as its relationship with the eyestalk. It was found that the injection of melatonin significantly increased the limb regeneration rate compared with the saline group (P < 0.05. The qRT-PCR results of growth-related genes showed that the level of EcR-mRNA (ecdysteroid receptor and Chi-mRNA (chitinase expression was significantly increased following the melatonin injection, while the expression of MIH-mRNA (molt-inhibiting hormone was significantly decreased (P < 0.05. Melatonin significantly increased lipase activity (P < 0.05. We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment, whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group (P < 0.05. Furthermore, the results of serum immune and antioxidant capacity revealed that melatonin significantly increased the total hemocyte counts (THC, hemocyanin content, total antioxidant capacity (T-AOC, acid phosphatase (ACP, and glutathione peroxidase activity (GSH-Px, whereas the immune-related parameters were significantly decreased in eyestalk-ablated crabs (P < 0.05. Therefore, these findings indicate that melatonin exerts a protective effect on organism injury, which could promote limb regeneration by up-regulating the expression of growth-related genes, improve digestive enzyme activity, and strengthen the immune response, particularly antioxidant capacity.

  6. 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.

  7. Mechanistic Insights on the Reductive Dehydroxylation Pathway for the Biosynthesis of Isoprenoids Promoted by the IspH Enzyme

    KAUST Repository

    Abdel-Azeim, Safwat

    2015-06-22

    Here, we report an integrated quantum mechanics/molecular mechanics (QM/MM) study of the bio-organometallic reaction pathway of the 2H+/2e- reduction of (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into the so called universal terpenoids precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), promoted by the IspH enzyme. Our results support the viability of the bio-organometallic pathway from rotation of the OH group of HMBPP away from the [Fe4S4] cluster at the core of the catalytic site, to be engaged in a H-bond with Glu126. This rotation is synchronous with π-coordination of the C2=C3 double bond of HMBPP to the apical Fe atom of the [Fe4S4] cluster. Dehydroxylation of HMBPP is triggered by a proton transfer from Glu126 to the OH group of HMBPP. The reaction pathway is completed by competitive proton transfer from the terminal phosphate group to the C2 or C4 atom of HMBPP.

  8. The major-effect quantitative trait locus CsARN6.1 encodes an AAA ATPase domain-containing protein that is associated with waterlogging stress tolerance by promoting adventitious root formation.

    Science.gov (United States)

    Xu, Xuewen; Ji, Jing; Xu, Qiang; Qi, Xiaohua; Weng, Yiqun; Chen, Xuehao

    2018-03-01

    In plants, the formation of hypocotyl-derived adventitious roots (ARs) is an important morphological acclimation to waterlogging stress; however, its genetic basis remains fragmentary. Here, through combined use of bulked segregant analysis-based whole-genome sequencing, SNP haplotyping and fine genetic mapping, we identified a candidate gene for a major-effect QTL, ARN6.1, that was responsible for waterlogging tolerance due to increased AR formation in the cucumber line Zaoer-N. Through multiple lines of evidence, we show that CsARN6.1 is the most possible candidate for ARN6.1 which encodes an AAA ATPase. The increased formation of ARs under waterlogging in Zaoer-N could be attributed to a non-synonymous SNP in the coiled-coil domain region of this gene. CsARN6.1 increases the number of ARs via its ATPase activity. Ectopic expression of CsARN6.1 in Arabidopsis resulted in better rooting ability and lateral root development in transgenic plants. Transgenic cucumber expressing the CsARN6.1 Asp allele from Zaoer-N exhibited a significant increase in number of ARs compared with the wild type expressing the allele from Pepino under waterlogging conditions. Taken together, these data support that the AAA ATPase gene CsARN6.1 has an important role in increasing cucumber AR formation and waterlogging tolerance. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  9. 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

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

    Science.gov (United States)

    Hassan, Husain; Abeer, Ali

    2014-01-01

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

  11. Quantitative measurement of membrane Na+-K+ ATPase activity using thallium-201: comparison with rubidium-86

    International Nuclear Information System (INIS)

    Lee, Jae Tae; Shon, Sang Kyun; Lee, Kyu Bo; Lee, In Kyu

    1998-01-01

    Na + -K + ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristics of Tl-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na + -K + ATPase activity using Tl-201 and compare with that using Rb-86. Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na + -K + ATPase activity. Na + -K + ATPase activity was measured as a percentage decrease in cellular uptake of Tl-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Na + -K + ATPase activity measured with Tl-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50-70% or 28% increase from baseline activity, respectively. These results suggests that Tl-201 could replace Rb-86 in measurement of ouabain sensitive Na + -K + ATPase activity in vitro. High level of glucose concentration decreased cellular Na + -K + ATPase activity, but insulin or PMA increased it

  12. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

    Science.gov (United States)

    Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

    2016-01-26

    The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology. Copyright © 2016, American Association for the Advancement of Science.

  13. Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

    Science.gov (United States)

    Beil, W.; Sewing, K. F.

    1984-01-01

    The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed. PMID:6146367

  14. 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.

  15. 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.

  16. 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.

  17. Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

    NARCIS (Netherlands)

    Wolvetang, E. J.; Wanders, R. J.; Schutgens, R. B.; Berden, J. A.; Tager, J. M.

    1990-01-01

    Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum

  18. 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.

  19. 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...

  20. THE EFFECT OF NIMODIPINE ON COCHLEAR POTENTIALS AND NA+/K+-ATPASE ACTIVITY IN NORMAL AND HYDROPIC COCHLEAS OF THE ALBINO GUINEA-PIG

    NARCIS (Netherlands)

    VANBENTHEM, PPG; KLIS, SFL; ALBERS, FWJ; DEWILDT, DJ; VELDMAN, JE; HUIZING, EH; SMOORENBURG, GF

    1994-01-01

    In experimental endolymphatic hydrops (EEH) a decrease in the endocochlear potential (EP) has been reported and is thought to be due to decreased activity of the enzyme Na+/K+-ATPase in the stria vascularis. By stimulating Na+/K+-ATPase, the EP, and thereby cochlear function as a whole, might be

  1. 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.

  2. 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.

  3. Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase.

    Science.gov (United States)

    Ahrens, M L

    1981-04-06

    In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.

  4. 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.

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

    Science.gov (United States)

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

    1990-01-01

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

  6. Genetic variants of methyl metabolizing enzymes and epigenetic regulators: Associations with promoter CpG island hypermethylation in colorectal cancer

    NARCIS (Netherlands)

    Vogel, S. de; Wouters, K.A.D.; Gottschalk, R.W.H.; Schooten, F.J. van; Goeij, A.F.P.M. de; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den; Weijenberg, M.P.; Engeland, M. van

    2009-01-01

    Aberrant DNA methylation affects carcinogenesis of colorectal cancer. Folate metabolizing enzymes may influence the bioavailability of methyl groups, whereas DNA and histone methyltransferases are involved in epigenetic regulation of gene expression. We studied associations of genetic variants of

  7. The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

    Science.gov (United States)

    Gahura, Ondřej; Šubrtová, Karolína; Váchová, Hana; Panicucci, Brian; Fearnley, Ian M; Harbour, Michael E; Walker, John E; Zíková, Alena

    2018-02-01

    The F-ATPases (also called the F 1 F o -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F 1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F 1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F 1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F 1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F 1 domain. These unique features of the F 1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F 1 -ATPase complex is not strictly conserved in eukaryotes. © 2017 Federation of European Biochemical Societies.

  8. A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

    Science.gov (United States)

    Pöhler, Robert; Krahn, Jan H; van den Boom, Johannes; Dobrynin, Grzegorz; Kaschani, Farnusch; Eggenweiler, Hans-Michael; Zenke, Frank T; Kaiser, Markus; Meyer, Hemmo

    2018-02-05

    AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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

  10. 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.

  11. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    International Nuclear Information System (INIS)

    Rocafull, Miguel A.; Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H + ), have been reported. They include reticulum and plasma-membrane Ca 2+ -ATPases, Na + /K + -ATPase and H + /K + -ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K + -ATPase α1-isoform, H + /K + -ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K + -ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  12. 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

  13. 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

  14. 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.

  15. Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

    Directory of Open Access Journals (Sweden)

    H.L. Santos

    2002-03-01

    Full Text Available SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM and low (K0.5 = 1.4 mM affinity sites for ATP, with negative cooperativity. Ouabain (5 mM, oligomycin (1 µg/ml and sodium vanadate (3 µM inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß2 association.

  16. Milrinone and thyroid hormone stimulate myocardial membrane Ca2+-ATPase activity and share structural homologies.

    Science.gov (United States)

    Mylotte, K M; Cody, V; Davis, P J; Davis, F B; Blas, S D; Schoenl, M

    1985-01-01

    We have recently shown that thyroid hormone in physiological concentrations stimulates sarcolemma-enriched rabbit-myocardial-membrane Ca2+-ATPase in vitro. In this study, milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1H)-one], a cardiac inotropic agent, was thyromimetic in the same system. At clinically achievable concentrations (50-500 nM), milrinone significantly stimulated membrane Ca2+-ATPase in vitro. This action was antagonized by W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an agent that also blocks thyroid hormone action on the Ca2+-ATPase, at concentrations as low as 5 microM. Progressive additions of milrinone to membranes incubated with a fixed concentration of thyroxine (0.10 nM) or triiodothyronine resulted in a progressive obliteration of the thyroid hormone effect on Ca2+-ATPase. Amrinone [5-amino-(3,4'-bipyridin)-6(1H)-one], the parent bipyridine of milrinone, had no effect on myocardial Ca2+-ATPase activity. X-ray crystallographic analysis of milrinone and amrinone revealed structural homologies between the phenolic ring of thyroxine and the substituted ring of milrinone, whereas amrinone did not share these homologies. The mechanism(s) of the inotropic actions of thyroxine and of milrinone is not clearly understood, but these observations implicate Ca2+-ATPase, a calcium pump-associated enzyme, as one mediator of the effects on the heart of these two compounds. PMID:2933747

  17. 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.

  18. Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

    International Nuclear Information System (INIS)

    Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

    1987-01-01

    To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

  19. 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...

  20. Search for the ouabain-binding site of Na,K-ATPase.

    NARCIS (Netherlands)

    Qiu, L.Y.

    2007-01-01

    Na,K-ATPase is an integral membrane protein found in almost all plasma membranes of higher eukaryotic cells. It maintains the electrochemical gradients present across the plasma membrane of the cells by catalyzing ATP-dependent transport of sodium and potassium ions. This enzyme is composed of two

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

    Science.gov (United States)

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

    2013-05-31

    Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography. Copyright © 2013 The Author. Published by Elsevier Inc. All rights reserved.

  2. Control analysis of the dependence of Escherichia coli physiology on the H+ -ATPase

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole; Westerhoff, Hans V.

    1993-01-01

    The H+-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and deter...

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

    NARCIS (Netherlands)

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

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

  4. The ntp operon encoding the Na+V-ATPase of the thermophile Caloramator fervidus

    NARCIS (Netherlands)

    Ubbink-Kok, Trees; Nijland, Jeroen; Slotboom, Dirk-Jan; Lolkema, Juke S.

    2006-01-01

    The V-type ATPase of the thermophile Caloramator fervidus is an ATP-driven Na+ pump. The nucleotide sequence of the ntpFIKECGABD operon containing the structural genes coding for the nine subunits of the enzyme complex was determined. The identity of the proteins in two pairs of subunits (D, E and

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

    Science.gov (United States)

    Moriyama, Y; Nelson, N

    1987-07-05

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

  6. 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.

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

    Science.gov (United States)

    Bredeston, Luis M; González Flecha, F Luis

    2016-07-01

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

  8. Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T.; Rubinstein, John L.

    2016-01-01

    Electron cryomicroscopy (cryo-EM) has significantly advanced our understanding of molecular structure in biology. Recent innovations in both hardware and software have made cryo-EM a viable alternative for targets that are not amenable to x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Cryo-EM has even become the method of choice in some situations where x-ray crystallography and NMR spectroscopy are possible but where cryo-EM can determine structures at higher resolution or with less time or effort. Rotary adenosine triphosphatases (ATPases) are crucial to the maintenance of cellular homeostasis. These enzymes couple the synthesis or hydrolysis of adenosine triphosphate to the use or production of a transmembrane electrochemical ion gradient, respectively. However, the membrane-embedded nature and conformational heterogeneity of intact rotary ATPases have prevented their high-resolution structural analysis to date. Recent application of cryo-EM methods to the different types of rotary ATPase has led to sudden advances in understanding the structure and function of these enzymes, revealing significant conformational heterogeneity and characteristic transmembrane α helices that are highly tilted with respect to the membrane. In this Review, we will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases. PMID:27532044

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Formation of oriented membrane multilayers of Na/K-ATPase

    International Nuclear Information System (INIS)

    Pachence, J.M.; Knott, R.; Edelman, I.S.; Schoenborn, B.P.; Wallace, B.A.

    1982-01-01

    The isolated membrane-bound enzyme retains its ouabain-sensitive ATP hydrolysis activity, and produces ATP-dependent Na + and K + fluxes when incorporated into phospholipid vesicles. The ultimate goal of this work is to determine its low resolution structure using both X-ray and neutron diffraction. A number of methods were used to impart lamellar stacking order to highly purified pig Na/K-ATPase membranes. Upon partial dehydration, x-ray diffraction from Na/K-ATPase membrane multilayers at 98% relative humidity yielded discrete reflections of 118 A periodicity, diffracting to 1/14.8 A -1 , additionally, continuous diffraction to 1/10 A -1 was obtained. Subjecting the membrane multilayers to high magnetic fields improved the quality of the lamellar diffraction dramatically. Neutron diffraction studies of the partially dehydrated Na/K-ATPase membrane multilayers detected a mosaic spread of 2 0 when the samples were subjected to a magnetic field of 5 Tesla perpendicular to the membrane surface; the reflections were narrower than the camera line width; hence, the lattice disorder has also decreased significantly, although only four orders were measured

  14. Long-Range Effects of Na(+) Binding in Na,K-ATPase Reported by ATP.

    Science.gov (United States)

    Middleton, David A; Fedosova, Natalya U; Esmann, Mikael

    2015-12-01

    This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.

  15. Enzyme Production and Nitrogen Fixation by Free, Immobilized and Coimmobilized Inoculants of Trichoderma harzianum and Azospirillum brasilense and Their Possible Role in Growth Promotion of Tomato

    Directory of Open Access Journals (Sweden)

    Momein H. El-Katatny

    2010-01-01

    Full Text Available A plant growth-promoting rhizobacterium (Azospirillum brasilense strain Az and a biocontrol fungus (Trichoderma harzianum strain T24 have been evaluated for their individual and combined production of hydrolytic enzymes, nitrogen fixation and their possible role in growth promotion of tomato seedlings. The studied organisms were inoculated as free or calcium alginate-encapsulated cells. All freshly prepared macrobeads showed high encapsulation capacity (EC/% of inocula compared with dry macrobeads. Results of enzyme production did not exhibit consistent pattern of the effect of encapsulation process on enzyme production. Beads entrapping bacterial and/or fungal cells were used successfully in 3 repeated cycles in the presence of fresh sterile culture medium in each growth cycle. Enzyme production by immobilized bacterial and/or fungal cells increased as the growth cycles were repeated. Co-culturing of A. brasilense with T. harzianum (free or immobilized in semisolid nitrogen deficient medium (N-free medium enabled A. brasilense to fix nitrogen on pectin, chitin and carboxymethyl cellulose. The activity of nitrogen fixation by A. brasilense in the case of single and combined cultures with Trichoderma (using dry encapsulated beads into the sterile soil increased with the addition of carbon source. Most of inoculations with free or alginate macrobead formulations of T. harzianum and/or A. brasilense showed significant increase in the growth parameters of tomato seedlings. The root system grew more profusely in the case of all seeds treated with A. brasilense. The growth parameters of Az/T24-treated seeds using dry coimmobilized macrobeads were higher than those of the untreated control. Moreover, the effect was improved significantly in soil enriched with different C sources. Enhanced tomato seedling growth after the co-inoculation could be due to the synergistic effect of both Trichoderma and Azospirillum. Finally, co-inoculation with Azospirillum

  16. Binding of the Inhibitor Protein IF1 to Bovine F1-ATPase

    Science.gov (United States)

    Bason, John V.; Runswick, Michael J.; Fearnley, Ian M.; Walker, John E.

    2011-01-01

    In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the α-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF1 and the C-terminal domains of the βDP-subunit and βTP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the βDP-subunit. Several conserved charged amino acids in the long α-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme. PMID:21192948

  17. Structure and Function of Cu(I)- and Zn(II)-ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg; Grønberg, Christina; Autzen, Henriette Elisabeth

    2015-01-01

    Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular...... membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing...

  18. A cytotoxic Petiveria alliacea dry extract induces ATP depletion and decreases β-F1-ATPase expression in breast cancer cells and promotes survival in tumor-bearing mice

    Directory of Open Access Journals (Sweden)

    John F. Hernández

    Full Text Available Abstract Metabolic plasticity in cancer cells assures cell survival and cell proliferation under variable levels of oxygen and nutrients. Therefore, new anticancer treatments endeavor to target such plasticity by modifying main metabolic pathways as glycolysis or oxidative phosphorylation. In American traditional medicine Petiveria alliacea L., Phytolaccacea, leaf extracts have been used for leukemia and breast cancer treatments. Herein, we study cytotoxicity and antitumoral effects of P. alliacea extract in tumor/non-tumorigenic cell lines and murine breast cancer model. Breast cancer cells treated with P. alliacea dry extract showed reduction in β-F1-ATPase expression, glycolytic flux triggering diminished intracellular ATP levels, mitochondrial basal respiration and oxygen consumption. Consequently, a decline in cell proliferation was observed in conventional and three-dimension spheres breast cancer cells culture. Additionally, in vivo treatment of BALB/c mice transplanted with the murine breast cancer TS/A tumor showed that P. alliacea extract via i.p. decreases the primary tumor growth and increases survival in the TS/A model.

  19. 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

  20. Critical role of γ-phosphate in structural transition of Na,K-ATPase upon ATP binding

    Science.gov (United States)

    Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Klimanova, Elizaveta A.; Dergousova, Elena A.; Lopina, Olga D.; Makarov, Alexander A.

    2014-06-01

    Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that β-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while γ-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the ``E1-open'' to ``E1-closed'' conformation ready for phosphorylation.

  1. Diallyl tetrasulfide improves cadmium induced alterations of acetylcholinesterase, ATPases and oxidative stress in brain of rats

    International Nuclear Information System (INIS)

    Pari, Leelavinothan; Murugavel, Ponnusamy

    2007-01-01

    Cadmium (Cd) is a neurotoxic metal, which induces oxidative stress and membrane disturbances in nerve system. The garlic compound diallyl tetrasulfide (DTS) has the cytoprotective and antioxidant activity against Cd induced toxicity. The present study was carried out to investigate the efficacy of DTS in protecting the Cd induced changes in the activity of acetylcholinesterase (AChE), membrane bound enzymes, lipid peroxidation (LPO) and antioxidant status in the brain of rats. In rats exposed to Cd (3 mg/kg/day subcutaneously) for 3 weeks, a significant (P + K + -ATPase, Mg 2+ -ATPase and Ca 2+ -ATPase) were observed in brain tissue. Oral administration of DTS (40 mg/kg/day) with Cd significantly (P < 0.05) diminished the levels of LPO and protein carbonyls and significantly (P < 0.05) increased the activities of ATPases, antioxidant enzymes, GSH and TSH in brain. These results indicate that DTS attenuate the LPO and alteration of antioxidant and membrane bound enzymes in Cd exposed rats, which suggest that DTS protects the brain function from toxic effects of Cd

  2. [The calix[4]arene C-107 is highly effective supramolecular inhibitor of the Na+,K(+)-ATPase of plasma membranes].

    Science.gov (United States)

    Bevza, O V; Veklich, T O; Shkrabak, O A; Rodik, R V; Kal'chenko, V I; Kosterin, S O

    2013-01-01

    The inhibition of the Na+,K(+)-ATPase activity of the myometrium cell plasma membranes with calixarene C-107 (5,17-diamino(2-pyridyl) methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) was investigated. It has been shown that calixarene C-107 reduced the Na+,K(+)-ATPase activity more efficiently than ouabain did, while it did not practically influence the "basal" Mg(2+)-ATPase activity of the same membrane. The magnitude of the cofficient of inhibition I0.5 was 33 +/- 4 nM, Hill coefficient was 0.38 +/- 0.06. The model calixarene C-150--the calixarene "scaffold" (26,28-dihydroxy-25,27-dipropoxycalix[4]arene), and the model compound M-3 (4-hydroxyaniline(2-pyridine)methylphosphonic acid)--a fragment of the calixarene C-107, had practically no influence on the enzymatic activity of Na+,K(+)-ATPase and Mg(2+)-ATPase. We carried out the computer simulation of interaction of calixarenes C-107 and the mentioned model compound with ligand binding sites of the Na+,K(+)-ATPase of plasma membrane and structure foundation of their intermolecular interaction was found out. The participation of hydrogen, hydrophobic, electrostatic and pi-pi (stacking) interaction between calixarene and enzyme aminoacid residues, some of which are located near the active center of Na+,K(+)-ATPase, was discussed.

  3. Protective Effect of Prosopis cineraria Against N-Nitrosodiethylamine Induced Liver Tumor by Modulating Membrane Bound Enzymes and Glycoproteins

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    Naina Mohamed Pakkir Maideen

    2012-06-01

    Full Text Available Purpose: The objective of the present study was to evaluate the protective effect of methanol extract of Prosopis cineraria (MPC against N-nitrosodiethylamine (DEN, 200mg/kg induced Phenobarbital promoted experimental liver tumors in male Wistar rats. Methods: The rats were divided into four groups, each group consisting of six animals. Group 1 served as control animals. Liver tumor was induced in group 2, 3, and 4 and Group 3 animals received MPC 200mg/kg and Group 4 animals received MPC 400mg/kg. Results: Administration of DEN has brought down the levels of membrane bound enzymes like Na+/ K+ ATPase, Mg2+ ATPase and Ca2+ATPase which were later found to be increased by the administration of Prosopis cineraria (200 and 400mg/kg in dose dependent manner. The MPC extract also suppressed the levels of glycoproteins like Hexose, Hexosamine and Sialic acid when compared to liver tumor bearing animals. Conclusions: Our study suggests that MPC may extend its protective role by modulating the levels of membrane bound enzymes and suppressing glycoprotein levels.

  4. Monoclonal antibody localization of Na+-K+-ATPase in the exocrine pancreas and parotid of the dog

    International Nuclear Information System (INIS)

    Smith, Z.D.J.; Caplan, M.J.; Forbush, B. III; Jamieson, J.D.

    1987-01-01

    A monoclonal antibody specific to the β-subunit of the canine 125 I-labeled-Na + -K + -ATPase has been characterized and used to directly localize the enzyme in thin frozen sections of dog pancreas and parotid. The antibody, 7-2M, recognizes only the β-subunit of the sodium pump as determined by immunoprecipitation and immunoblot and is not directed against an oligosaccharide determinant. 7-2M immunolocalizes to the same cellular and subcellular domains of renal tubular cells as do other, previously characterized, antibodies directed to the α-subunit of the sodium pump. In the pancreas the preponderance of the Na + -K + -ATPase is found on the basolateral membranes of centroacinar and intralobular duct cells. Interlobular duct cells also express a large component of basolaterally located enzyme, although comparatively little pump is seen on acinar cells. In the parotid a large amount of Na + -K + -ATPase is seen on the striated cut cells, with high levels also noted on cells of the intercalated ducts and serous demilunes. Again the acinar cells show comparatively low levels of Na + -K + -ATPase. In no instance is Na + -K + -ATPase found on the apical membranes of pancreas or parotid cells. These data suggest that Na + -K + -ATPase, located on the basolateral plasmalemma of duct-derived cells, may be involved in water and electrolyte secretion from the pancreas and parotid

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

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    Soontharapirakkul Kanteera

    2010-08-01

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

  6. Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids.

    Science.gov (United States)

    Cornelius, Flemming; Mahmmoud, Yasser A; Toyoshima, Chikashi

    2011-08-26

    The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.

  7. Isolation, crystallization and crystal structure determination of bovine kidney Na(+),K(+)-ATPase.

    Science.gov (United States)

    Gregersen, Jonas Lindholt; Mattle, Daniel; Fedosova, Natalya U; Nissen, Poul; Reinhard, Linda

    2016-04-01

    Na(+),K(+)-ATPase is responsible for the transport of Na(+) and K(+) across the plasma membrane in animal cells, thereby sustaining vital electrochemical gradients that energize channels and secondary transporters. The crystal structure of Na(+),K(+)-ATPase has previously been elucidated using the enzyme from native sources such as porcine kidney and shark rectal gland. Here, the isolation, crystallization and first structure determination of bovine kidney Na(+),K(+)-ATPase in a high-affinity E2-BeF3(-)-ouabain complex with bound magnesium are described. Crystals belonging to the orthorhombic space group C2221 with one molecule in the asymmetric unit exhibited anisotropic diffraction to a resolution of 3.7 Å with full completeness to a resolution of 4.2 Å. The structure was determined by molecular replacement, revealing unbiased electron-density features for bound BeF3(-), ouabain and Mg(2+) ions.

  8. Cellular localization of Na(+), K(+)-ATPase in the mammalian vestibular system

    Science.gov (United States)

    Kerr, T. P.

    1984-01-01

    Two different, but complementary, procedures for cellular localization of Na+, K+-ATPase in the guinea pig vestibular system were employed. One of these techniques, devised by Stirling, depends upon the well documented ability of the specific inhibitor ouabain to bind selectively to Na+,K+-ATPase, blocking catalytic activity. Microdisected vestibular tissues are incubated with tritium-labelled (3H-) ouabain, and regions with a high concentration of Na+,K+-ATPase are subsequently identified by light microscope autoradiography. A second method, originated by Ernst, detects inorganic phosphate released from an artificial substrate (nitrophenyl phosphate) by catalytic activity of the enzyme. In the presence of strontium ion, phosphate is precipitated near regions of high activity, then converted to a product which may finally be visualized in the electron microscope. This cytochemical enzymatic reaction is inhibited by ouabain.

  9. H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

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    Ulfah Lutfiah

    2015-11-01

    Full Text Available Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA. This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2, and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159, and drastically lowered from the third to seventh day (0.289; 0.108; 0.062. Specific activities of H,K-ATPase was slightly higher in the first day (0.765, then was lowered in the third (0.685 and fifth day (0.655, and was higher in the seventh day (0.884. The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343. Specific activities of total CA was low in the first day (0.083, and was higher from the third to seventh day (0.111; 0.136; 0.144.Conclusion: In hypoxia

  10. 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

  11. 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.

  12. The ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering

    Science.gov (United States)

    Çamdere, Gamze; Guacci, Vincent; Stricklin, Jeremiah; Koshland, Douglas

    2015-01-01

    Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin’s DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition. DOI: http://dx.doi.org/10.7554/eLife.11315.001 PMID:26583750

  13. Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria.

    Science.gov (United States)

    Pagliarani, Alessandra; Bandiera, Patrizia; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Nesci, Salvatore; Borgatti, Anna Rosa

    2008-06-01

    Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

  14. 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

  15. Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman.

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    Alexander V Chibalin

    Full Text Available Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV while the activity of the α1 isoform decreased (-4.4 mV. Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM, measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63 and Ser(68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.

  16. 31P NMR Spectroscopy Revealed Adenylate kinase-like Activity and Phosphotransferase-like Activity from F1-ATPase of Escherichia coli

    International Nuclear Information System (INIS)

    Kim, Hyun Won

    2011-01-01

    Adenylate kinase-like activity and phosphotransferase-like activity from F 1 -ATPase of Escherichia coli was revealed by 31 P NMR spectroscopy. Incubation of F 1 -ATPase with ADP in the presence of Mg 2+ shows the appearance of 31 P resonances from AMP and Pi, suggesting generation of AMP and ATP by adenylate kinase-like activity and the subsequent hydrolysis to Pi. Incubation of F1-ATPase with ADP in the presence of methanol shows additional peak from methyl phosphate, suggesting phosphotransferase-like activity of F 1 -ATPase. Both adenylate kinase-like activity and phosphotransferase-like activity has not been reported from F 1 -ATPase of Escherichia coli. 31 P NMR could be a valuable tool for the investigation of phosphorous related enzyme

  17. Changes in acetylcholinesterase, Na+,K+-ATPase, and Mg2+-ATPase activities in the frontal cortex and the hippocampus of hyper- and hypothyroid adult rats.

    Science.gov (United States)

    Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

    2007-08-01

    The thyroid hormones (THs) are crucial determinants of normal development and metabolism, especially in the central nervous system. The metabolic rate is known to increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-adenosinetriphosphatase (ATPase) in the frontal cortex and the hippocampus of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, and hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. A region-specific behavior was observed concerning the examined enzyme activities in hyper- and hypothyroidism. In hyperthyroidism, AChE activity was significantly increased only in the hippocampus (+22%), whereas Na+,K+-ATPase activity was significantly decreased in the hyperthyroid rat hippocampus (-47%) and remained unchanged in the frontal cortex. In hypothyroidism, AChE activity was significantly decreased in the frontal cortex (-23%) and increased in the hippocampus (+21%). Na+,K+-ATPase activity was significantly decreased in both the frontal cortex (-35%) and the hippocampus (-43%) of hypothyroid rats. Mg2+-ATPase remained unchanged in the regions of both hyper- and hypothyroid rat brains. Our data revealed that THs affect the examined adult rat brain parameters in a region- and state-specific way. The TH-reduced Na+,K+-ATPase activity may increase the synaptic acetylcholine release and, thus, modulate AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems in the examined brain regions.

  18. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    International Nuclear Information System (INIS)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

    1990-01-01

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

  19. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  20. Heat death in the crayfish Austropotamobius pallipes: thermal inactivation of muscle membrane-bound ATPases in warm and cold adapted animals

    Energy Technology Data Exchange (ETDEWEB)

    Gladwell, R T

    1976-01-01

    The thermal sensitivity of the membrane-bound Mg/sup 2 +/ and Na/sup +/ + K/sup +/ + Mg/sup 2 +/ ATPases from the abdominal flexor muscles of 10 and 25/sup 0/C acclimated animals was investigated. The Mg/sup 2 +/ ATPase was inactivated by milder heat treatments than the Na/sup +/ + K/sup +/ + Mg/sup 2 +/ ATPase. The effect of high lethal temperatures on the Mg/sup 2 +/ ATPase was dependent on the previous thermal history of the animal, the enzyme preparations from 10/sup 0/C acclimated animals being more sensitive than those from 25/sup 0/C acclimated animals. The thermal sensitivity of the Na/sup +/ + K/sup +/ + Mg/sup 2 +/ ATPase was not altered by temperature acclimation. The change in the thermal sensitivity of the Mg/sup 2 +/ ATPase with the acclimation temperature of the whole animal was correlated with the survival times of 10 and 25/sup 0/C acclimated animals. The K/sub m/ and V/sub max/ of the ATPases was investigated and the K/sub m/ of both enzymes was found to decrease with acclimation of the whole animal to lower temperatures, so that enzyme/substrate affinity increased with cold acclimation. It was concluded that the inactivation of the muscle Mg/sup 2 +/ ATPase was the primary lesion of heat death in the crayfish, and that the changes in the kinetic properties of the ATPases were an important mechanism in the process of physiological temperature acclimation.

  1. Immobilization of Na,K-ATPase isolated from rat brain synaptic plasma membranes

    Directory of Open Access Journals (Sweden)

    ANICA HROVAT

    2002-12-01

    Full Text Available Rat brain Na,K-ATPase partially purified by SDS from synaptic plasma membranes (SPM was immobilized by adsorption on nitrocellulose (NC, polyvinylidene fluoride (PVDF and glass fiber (GF membranes. Partial SDS solubilization increased the enzyme activity by 40 %. With regard to the preservation of the enzyme activity, nitrocellulose was shown to be the optimal support for the immobilization. The enzyme showed the highest percentage activity (14 % after 30 min of SPM adsorption, at 20°C under the vaccum, with 25 mg of proteins per NC disc filter. In addition, adsorption on NC stabilizes the Na,K-ATPase, since the activity was substantial 72 h after adsorption at 20°C. After adsorption, the sensitivity of the enzyme to HgCl2and CdCll2 inhibition was higher. The results show that immobilized Na,K-ATPase SPM can be used as a practical model for the detection of metal ions in different samples.

  2. 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.

  3. Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

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    Nimgampalle Mallikarjuna

    2016-12-01

    Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20% in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration.

  4. The influence of temperature on the distribution and intensity of the reaction product in rat muscle fibers obtained with the histochemical method for myosin ATPase

    DEFF Research Database (Denmark)

    Kirkeby, S; Tuxen, A

    1989-01-01

    The influence of temperature in the incubation medium on the localization and intensity of myosin ATPase was investigated in striated muscles from the rat using a conventional histochemical technique. It was found that the enzyme reaction was temperature-dependent since the activity in some fibers...... was raised and in others was depressed by alteration of the incubation temperature. There was no obvious correlation between the temperature sensitivity of ATPase in the muscle fibers and their activity for succinic dehydrogenase. It is proposed that the histochemical method for myosin ATPase can be used...

  5. 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

  6. Inhibition of the gut enzyme intestinal alkaline phosphatase may explain how aspartame promotes glucose intolerance and obesity in mice.

    Science.gov (United States)

    Gul, Sarah S; Hamilton, A Rebecca L; Munoz, Alexander R; Phupitakphol, Tanit; Liu, Wei; Hyoju, Sanjiv K; Economopoulos, Konstantinos P; Morrison, Sara; Hu, Dong; Zhang, Weifeng; Gharedaghi, Mohammad Hadi; Huo, Haizhong; Hamarneh, Sulaiman R; Hodin, Richard A

    2017-01-01

    Diet soda consumption has not been associated with tangible weight loss. Aspartame (ASP) commonly substitutes sugar and one of its breakdown products is phenylalanine (PHE), a known inhibitor of intestinal alkaline phosphatase (IAP), a gut enzyme shown to prevent metabolic syndrome in mice. We hypothesized that ASP consumption might contribute to the development of metabolic syndrome based on PHE's inhibition of endogenous IAP. The design of the study was such that for the in vitro model, IAP was added to diet and regular soda, and IAP activity was measured. For the acute model, a closed bowel loop was created in mice. ASP or water was instilled into it and IAP activity was measured. For the chronic model, mice were fed chow or high-fat diet (HFD) with/without ASP in the drinking water for 18 weeks. The results were that for the in vitro study, IAP activity was lower (p < 0.05) in solutions containing ASP compared with controls. For the acute model, endogenous IAP activity was reduced by 50% in the ASP group compared with controls (0.2 ± 0.03 vs 0.4 ± 0.24) (p = 0.02). For the chronic model, mice in the HFD + ASP group gained more weight compared with the HFD + water group (48.1 ± 1.6 vs 42.4 ± 3.1, p = 0.0001). Significant difference in glucose intolerance between the HFD ± ASP groups (53 913 ± 4000.58 (mg·min)/dL vs 42 003.75 ± 5331.61 (mg·min)/dL, respectively, p = 0.02). Fasting glucose and serum tumor necrosis factor-alpha levels were significantly higher in the HFD + ASP group (1.23- and 0.87-fold increases, respectively, p = 0.006 and p = 0.01). In conclusion, endogenous IAP's protective effects in regard to the metabolic syndrome may be inhibited by PHE, a metabolite of ASP, perhaps explaining the lack of expected weight loss and metabolic improvements associated with diet drinks.

  7. “Oxygen sensing” by Na,K-ATPase: these miraculous thiols

    Directory of Open Access Journals (Sweden)

    Anna Bogdanova

    2016-08-01

    Full Text Available Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O2 binding sites its oxygen-sensitivity is mediated by a variety of redox-sensitive modifications including S-glutathionylation, S-nitrosylation and redox-sensitive phosphorylation. This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidised glutathione are the signalling messengers that make the Na,K-ATPase oxygen-sensitive. This very ancient signalling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the optimal level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterise the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summery on (i the sources of free radical production in hypoxic cells, (ii localisation of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzymes to a variety of stimuli (hypoxia, receptors’ activation control of the enzyme activity (iii redox-sensitive regulatory phosphorylation, and (iv the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate

  8. Digitalis-induced cell signaling by the sodium pump: on the relation of Src to Na(+)/K(+)-ATPase.

    Science.gov (United States)

    Gable, Marjorie E; Abdallah, Simon L; Najjar, Sonia M; Liu, Lijun; Askari, Amir

    2014-04-18

    In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na(+)/K(+)-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na(+)/K(+)-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na(+)/K(+)-ATPase, Mg(2+), and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na(+)/K(+)-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na(+)/K(+)-ATPase under physiological conditions. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Temperature-specific inhibition of human red cell (Na/sup +//K/sup +/) ATPase by 2450-MHz microwave radiation

    Energy Technology Data Exchange (ETDEWEB)

    Allis, J.W.; Sinha-Robinson, B.L.

    1987-01-01

    The ATPase activity in human red blood cell membranes was investigated in vitro as a function of temperature and exposure to 2450-MHz (CW) microwave radiation. Assays were conducted spectrophotometrically during microwave exposure with a custom-made spectrophotometer-waveguide apparatus. Temperature profiles of total ATPase and Ca+2 ATPase (ouabain-inhibited) activity between 17 and 31 C were graphed as an Arrhenius plot. Each data set was fitted to two straight lines which intersected between 23 and 24 C. The difference between the total and Ca+2 ATPase activities, which represented the Na+/K+ ATPase activity, was also plotted and treated similarly to yield an intersection near 25 C. Exposure of membrane suspensions to a 6 W/kg dose rate at 1 C intervals between 23 and 27 C, resulted in an activity change only for the Na+/K+ ATPase at 25 C. The activity decreased by approximately 35% compared to sham-irradiated samples. An hypothesis based on the interaction of microwave radiation with enzyme structure during a conformational rearrangement is proposed as an explanation for the effect.

  10. Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs of Lymnaea stagnalis and Bithynia tentaculata (Mollusca)

    NARCIS (Netherlands)

    D. Zivkovic (Dana); R. Créton (Robbert); G. Zwaan (Gideon); W.C. de Bruijn (Wim); M.R. Dohmen (M.René)

    1990-01-01

    textabstractDuring extrusion of the first polar body in eggs of Lymnaea stagnalis and Bithynia tentaculata a localized Ca2+ /Mg2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity

  11. 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.

  12. 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.

  13. 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.

  14. Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase

    DEFF Research Database (Denmark)

    Ebbesson, Lars O E; Tipsmark, Christian K; Holmqvist, Bo

    2005-01-01

    We investigated the relationship between nitric oxide (NO) and Na(+),K(+)-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphor...

  15. Isoform-Specific Na,K-ATPase Alterations Precede Disuse-Induced Atrophy of Rat Soleus Muscle

    Directory of Open Access Journals (Sweden)

    Violetta V. Kravtsova

    2015-01-01

    Full Text Available This study examines the isoform-specific effects of short-term hindlimb suspension (HS on the Na,K-ATPase in rat soleus muscle. Rats were exposed to 24–72 h of HS and we analyzed the consequences on soleus muscle mass and contractile parameters; excitability and the resting membrane potential (RMP of muscle fibers; the electrogenic activity, protein, and mRNA content of the α1 and α2 Na,K-ATPase; the functional activity and plasma membrane localization of the α2 Na,K-ATPase. Our results indicate that 24–72 h of HS specifically decreases the electrogenic activity of the Na,K-ATPase α2 isozyme and the RMP of soleus muscle fibers. This decrease occurs prior to muscle atrophy or any change in contractile parameters. The α2 mRNA and protein content increased after 24 h of HS and returned to initial levels at 72 h; however, even the increased content was not able to restore α2 enzyme activity in the disused soleus muscle. There was no change in the membrane localization of α2 Na,K-ATPase. The α1 Na,K-ATPase electrogenic activity, protein and mRNA content did not change. Our findings suggest that skeletal muscle use is absolutely required for α2 Na,K-ATPase transport activity and provide the first evidence that Na,K-ATPase alterations precede HS-induced muscle atrophy.

  16. 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).

  17. In situ assembly states of (Na+,K+)-pump ATPase in human erythrocytes. Radiation target size analyses

    International Nuclear Information System (INIS)

    Hah, J.; Goldinger, J.M.; Jung, C.Y.

    1985-01-01

    The in situ assembly state of the (Na+,K+)-pump ATPase of human erythrocytes was studied by applying the classical target theory to radiation inactivation data of the ouabain-sensitive sodium efflux and ATP hydrolysis. Erythrocytes and their extensively washed white ghosts were irradiated at -45 to -50 degrees C with an increasing dose of 1.5-MeV electron beam, and after thawing, the Na+-pump flux and/or enzyme activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose, from which a radiation sensitive mass (target size) was calculated. When intact cells were used, the target sizes for the pump and for the ATPase activities were equal and approximately 620,000 daltons. The target size for the ATPase activity was reduced to approximately 320,000 daltons if the cells were pretreated with digitoxigenin. When ghosts were used, the target size for the ATPase activity was again approximately 320,000 daltons. Our target size measurements together with other information available in literature suggest that (Na+,K+)-pump ATPase may exist in human erythrocytes either as a tetramer of alpha beta or as a dimer of alpha beta in tight association with other protein mass, probably certain glycolytic enzymes, and that this tetrameric or heterocomplex association is dissociable by digitoxigenin treatment or by extensive wash during ghost preparation

  18. 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.

  19. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    Energy Technology Data Exchange (ETDEWEB)

    Rocafull, Miguel A., E-mail: mrocaful@ivic.ve [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca{sup 2+}, Na{sup +}, K{sup +} and H{sup +}), have been reported. They include reticulum and plasma-membrane Ca{sup 2+}-ATPases, Na{sup +}/K{sup +}-ATPase and H{sup +}/K{sup +}-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg{sup 2+}ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na{sup +}/K{sup +}-ATPase {alpha}1-isoform, H{sup +}/K{sup +}-ATPase {alpha}2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H{sup +}/K{sup +}-ATPase {alpha}2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

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

    International Nuclear Information System (INIS)

    Hayashi, M.; Katz, A.I.

    1987-01-01

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

  1. Crystals of Na(+)/K(+)-ATPase with bound cisplatin.

    Science.gov (United States)

    Huliciak, Miroslav; Reinhard, Linda; Laursen, Mette; Fedosova, Natalya; Nissen, Poul; Kubala, Martin

    2014-12-01

    Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na(+)/K(+)-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4 Å and 7.9 Å), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

    International Nuclear Information System (INIS)

    Morales-Ríos, Edgar; Montgomery, Martin G.; Leslie, Andrew G. W.; García-Trejo, José J.; Walker, John E.

    2015-01-01

    The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F 1 domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F 1 –ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized

  3. Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Ríos, Edgar; Montgomery, Martin G. [The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom); Leslie, Andrew G. W. [The Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH (United Kingdom); García-Trejo, José J. [Universidad Nacional Autónoma de México, Mexico City (Mexico); Walker, John E., E-mail: walker@mrc-mbu.cam.ac.uk [The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom)

    2015-09-23

    The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F{sub 1} domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F{sub 1}–ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-03

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

  6. 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.

  7. The influence of ionizing radiation on the structure of Ca-ATPase hydrophobic fragment of skeletal muscle sarcoplasmic reticulum

    International Nuclear Information System (INIS)

    Vojtsitskij, V.M.; Fedorov, A.N.; Lugovskoj, Eh.B.; Derzskaya, S.G.; Khizhnyak, S.V.; Kurskij, M.D.; Kucherenko, N.E.

    1990-01-01

    Early (1 and 24 h) after X-irradiation with a dose of 0.21 C/kg changes occurred in the acceptibility of the polypeptide chain parts of sarcoplasmic reticulum Ca-ATPase for the effect of trypsin. The analysis of the results of studying the structural and functional properties of a hydrophobic fragment of this enzyme in the control and after irradiation permitted to define the part of the Ca-ATPase polypeptide chain that provided ion selectivity of the fragment

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  9. Gill Na+, K+-ATPase activity in largemouth bass (Micropterus salmoides) inhabiting reservoirs contaminated with mercury

    International Nuclear Information System (INIS)

    Brundage, S.; Jagoe, C.H.; Shaw-Allen, P.

    1995-01-01

    Active transport of Na + and K + for osmoregulation in fish involves gill Na + , K + -ATPase, a membrane-bound enzyme powered by hydrolysis of ATP. Na + , K + -ATPase is inhibited by many dissolved metals including Al, Cd, Cu and Hg, resulting in ionoregulatory dysfunction. However, dissolved Hg concentrations are quite low in most aquatic systems, and dietary sources are the most important contributors to Hg burdens in fish. One recent study demonstrated relationships between muscle Hg concentration and gill Na + , K + -ATPase in a marine fish, suggesting that Hg accumulated via diet can affect osmoregulation. The authors tested for such a relationship in several age-classes of a freshwater fish (Micropterus salmoides) collected from three reservoirs. Fish from Par Pond and L Lake, on the USDOE Savannah River Site in South Carolina had relatively high Hg content: for Par Pond, muscle and liver ranged from 1.58--12.01 and 1.46--23.22 microg Hg/g dry mass, respectively, and for L Lake muscle and liver ranged from 3.11--5.16 and 1.28--12.59 microg Hg/g dry mass, respectively. Bass from an offsite location, Thurmond Lake, had significantly (P + , K + -ATPase activity was not evident

  10. Characteristics of a (Na+K+)-ATPase inhibitor in extracts of tea

    International Nuclear Information System (INIS)

    Sagnella, G.A.; MacGregor, G.A.

    1984-01-01

    Extracts of tea were examined for inhibitors of the sodium-potassium pump by investigating the effect of the extracts on 1) isolated preparations of (Na + -K + )-ATPase from hog brain and human blood cells; 2) the displacement of radioactive ouabain from its specific receptor on red blood cells, and 3) the uptake of radioactive rubidium in intact red blood cells. It has been found that extracts of tea were potent inhibitors of the purified hog brain (Na + -K + )-ATPase. However, the inhibition was not specific for the (Na + -K + )-ATPase and the extract of tea did not displace 3 H-ouabain in a specific ouabain-receptor assay. Additionally, the tea extracts displayed only a small inhibitory effect on the uptake of 86 Rb in intact red blood cells. These observations suggest that the material is not like digitalis and that, unlike cardiac glycosides, it may inhibit the activity of the (Na + -K + )-ATPase by interacting with the enzyme at intracellular sites

  11. 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.

  12. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    Science.gov (United States)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  13. 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...

  14. Enzyme 15-lipoxygenase 1 promotes hypoxia-inducible factor 1α turnover and reduces vascular endothelial growth factor expression: implications for angiogenesis

    International Nuclear Information System (INIS)

    Zhong, Hua; Wang, Ruoxiang; Kelavkar, Uddhav; Wang, Christopher Y; Simons, Jonathan

    2014-01-01

    Hypoxia-inducible factor 1α (HIF-1α) is the regulatory subunit of the heterodimeric HIF-1 that plays a critical role in transcriptional regulation of genes in angiogenesis and hypoxic adaptation, while fatty acid metabolism mediated by lipoxygenases has been implicated in a variety of pathogeneses, including cancers. In this study, we report that 15-lipoxygenase 1 (15-LO1), a key member of the lipoxygenase family, promotes HIF-1α ubiquitination and degradation. Altering the level of 15-LO1 yields inverse changes in HIF-1α and HIF-1 transcriptional activity, under both normoxia and hypoxia, and even in CoCl 2 -treated cells where HIF-1α has been artificially elevated. The antagonistic effect of 15-LO1 is mediated by the Pro 564 /hydroxylation/26S proteasome system, while both the enzymatic activity and the intracellular membrane-binding function of 15-LO1 appear to contribute to HIF-1α suppression. Our findings provide a novel mechanism for HIF-1α regulation, in which oxygen-dependent HIF-1 activity is modulated by an oxygen-insensitive lipid metabolic enzyme

  15. 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.

  16. 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.

  17. The use of lac-type promoters in control analysis

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Westerhoff, H. v.; Michelsen, Ole

    1993-01-01

    experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H+-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times...... the wild-type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level. We show that the introduction of a lac Y mutation facilitated...

  18. 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.

  19. 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...

  20. 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

  1. 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.

  2. Vanadate and phosphotransferases with special emphasis on ouabain/Na, K-ATPase interaction

    International Nuclear Information System (INIS)

    Hansen, O.

    1983-01-01

    A short introduction to the chemistry and possible physiological or toxicological role of vanadium is given. The +5 oxidation state, vanadate, is a very efficient inhibitor of several phosphotransferases and for that reason a prosperous tool in the study of such enzymes. Special emphasis is placed on studies with vanadate on the sodium pump. Vanadate appears to supplement ouabain as high affinity inhibitor of Na,K-ATPase. They are also complementary to one another since vanadate binds to the cytoplasmic aspect and ouabain to the extracellular side of the cell membrane, and moreover, they potentiate the binding of one another. The hypothesis that vanadate may act as a transition state analogue of phosphate seems supported by the observation that vanadate, like phosphate, is able to induce ouabain binding to Na,K-ATPase. The vanadate affinity is much higher than that of phosphate, however, and vanadate remains bound in a rather stable enzyme-vanadate-ouabain complex. Fluorescene studies indicate that different subspecies of an E 2 -conformation of the enzyme is obtained with vanadate and with ouabain. In molecular studies on Na,K-ATPase, e.g. in studies on the protein folding through the plasma-membrane, one can imagine that the simultaneous binding of an extracellular marker, ouabain, and of the intracellular marker, vanadate, may be most helpful tools. A proposed physiological regulatory role of vanadium on the pump activity seems less likely considering the simultaneous acceleration of K + -uptake which is probably due to adenylate cyclase activation. Vanadate seems more likely to have a pharmacological role as a cofactor for digitals binding to Na,K-ATPase. Finally, the vasoconstriction evoked by vanadate could indicate a pathophysiological role of the ion and vanadate could then become useful tool in experimental hypertension and uremia. (author)

  3. Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals.

    Science.gov (United States)

    Dalla, Safaa; Swarts, Herman G P; Koenderink, Jan B; Dobler, Susanne

    2013-12-01

    Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Changes in the corneal Na-K ATPase levels in eyes stored in moist chamber at 4°C

    Directory of Open Access Journals (Sweden)

    Devi B

    1996-01-01

    Full Text Available This report deals with a chronological measurement of Na-K ATPase enzyme activity in human and bovine corneas stored in a moist chamber at 4°C. Paired human and bovine eyes were sterilized by the standard eye bank procedure and stored up to 6 days. At the desired time, the corneal endothelium was assayed for Na-K ATPase activity. The protein content of each tissue sample was also determined. In a parallel set of experiments, the viability of identical stored corneas was determined by trypan blue and alizarin red staining technique, and morphometric analysis was done to quantify the extent of the corneal endothelial damage. The human corneas showed that there was a significant progressive decrease in the Na-K ATPase activity as the storage time increased. The decrease was related to morphological endothelial damage.

  5. Identification of the segment of the catalytic subunit of (Na+,K+)ATPase containing the digitalis binding site.

    Science.gov (United States)

    Rossi, B; Ponzio, G; Lazdunski, M

    1982-01-01

    Digitalis compounds that are extensively used in the treatment of cardiovascular disorders are known to bind specifically at the extracellular side of (Na+,K+)ATPase. We have recently reported the synthesis of [3H]p- nitrophenyltriazene -ouabain, a derivative of ouabain, which specifically alkylates the catalytic chain of the (Na+,K+)ATPase at a defined region of the sequence. The peptidic segment involved in the binding of digitalis to (Na+,K+)ATPase has been located after mild trypsin treatment of the labeled enzyme. In the presence of 100 mM KCl, tryptic fragmentation results in two peptide fragments of mol. wt. 58 000 and 41 000, respectively. The radioactive probe labeled only the 41 000 fragment indicating that the digitalis binding site is located on the 41 000 domain situated at the N-terminal part of the sequence of the alpha-subunit. Images Fig. 1. Fig. 3. PMID:6329711

  6. THE COMPARATIVE ANALYSIS OF THE ACTIVITY ASSAY METHODS FOR MG2+-DEPENDENT NA+/K+-ACTIVATED ATPASE IN ERYTHROCYTE MEMBRANES

    Directory of Open Access Journals (Sweden)

    Polina Anatolevna Petrova

    2017-12-01

    Full Text Available This review considers the methodological reasons for the wide range of results for the red blood cells Mg2+-dependent Na+/K+-ATPase activity described by different authors. We assert that the differences in the Na+/K+-ATPase activity obtained by the researchers are due to the methodological peculiarities associated with methods of obtaining and measurement of the enzyme activity, such as red blood cells separation and storage (centrifugation, concentration and composition of the lysing solution, time and temperature of hemolysis and freezing, as well as the peculiarities of methods for the quantitative determination of protein and inorganic phosphorus. On the basis of the literature data analysis we recommend that for the most accurate determination of the Na+/K+-ATPase activity it is better to use the chelator in the lysing buffer solution and Fiske-Subbarow and Lowry methods for the determination of inorganic phosphorus and quantitative protein content, respectively.

  7. Residues of organochlorine pesticides in fish, crab and sediment from El Temsah Lake, Suez Canal, Egypt and their effect on mitochondrial ATPase of the New Zealand white rabbit.

    Science.gov (United States)

    Ahmed, M T; Ismail, S M

    1991-01-01

    Residues of organochlorine pesticides were monitored in the muscles of Bolti fish Tilapia zillii, the crab Lupa pelagicus and sediment samples collected from El Temsah lake around Ismailia using gas liquid chromatography. The beta isomer of hexachlorocyclohexane (beta.HCH) was the most dominant compound detected in all samples, followed by P, P-DDE and P, P-DDT. Results showed the crab to contain higher concentrations of organochlorine in comparison to concentrations detected in fish muscles. The In-vitro effect of the residues extracted from fish, and crab on the mitochondrial brain and liver ATPase of the New Zealand white rabbit Orcytolagus cuniculus was also studied. Residues of organochlorine pesticides have induced activation in the ATPase enzyme system of both brain and liver. The mixtures of organochlorine residues of both fish and crab were able to activate liver ATPase more than brain ATPase. The present study was conducted to extrapolate possible effects incurred on man if consumed such food.

  8. Osmotic and thermal effects on in situ ATPase activity in permeabilized gill epithelial cells of the fish Gillichthys mirabilis

    Science.gov (United States)

    KÜLtz; Somero

    1995-01-01

    Long-jawed mudsuckers (Gillichthys mirabilis) were acclimated to sea water (SW) at 7 °C, SW at 26 °C or dilute sea water (DSW) at 26 °C for 5 months. Gill cells were isolated and the proportion of mitochondria-rich (MR) cells was determined. The number of cells harvested amounted to 4.7x10(7)±0.6x10(7) to 10.6x10(7)±1.1x10(7) and the yield was between 7.1x10(8)±0.6x10(8) and 10.7x10(8)±1.4x10(8) cells g-1 gill epithelial mass. Cell viability was 96.8±0.4 to 97.8±0.6 %. The number, size and volume of MR cells decreased significantly during DSW acclimation, but did not change during thermal acclimation. The protein content was not influenced by osmotic or thermal acclimation and ranged between 20.0±1.6 and 22.1±1.5 pg cell-1. Using a new method, which is based on the formation of plasma membrane channels by alamethicin, we were able to permeabilize gill cells. For the first time, the Na+/K+-ATPase and H+-ATPase activities of fish gills were determined in intact cells in situ. The activity of both ATPases was dependent on alamethicin concentration (optimum 100 µg mg-1 protein) and on preincubation time (optimum 10 min). The in situ activity of both ATPases was influenced by osmotic, but not thermal, acclimation. A positive linear correlation was found between in situ Na+/K+-ATPase activity and total MR cell volume. However, we show, for the first time, that a negative linear correlation exists between H+-ATPase activity and total MR cell volume, suggesting a localization of H+-ATPase in pavement cells. In permeabilized cells, the activity of both ATPases was 2.6­3.9 times higher than that of crude homogenates and 1.6­2.1 times higher than that of permeabilized homogenate vesicles. We hypothesize that in crude homogenates three-quarters of Na+/K+-ATPase and two-thirds of H+-ATPase activity are not detectable both because of a mixture of inside-out and right-side-out vesicles and because of the disruption of membrane and enzyme integrity.

  9. Changes of mitochondrial structure, ATPase and Ca2+ concentration in spermatogenic cells of mouse testes induced by low dose radiation

    International Nuclear Information System (INIS)

    Wang Zhicheng; Liu Shuchun; Li Pengwu; Kang Shunai; Liang Shuo; Zhao Gang; Gong Shouliang

    2009-01-01

    Objective: To observe the ultrastructure, ATPase activity and Ca 2+ concentration ([Ca 2+ ]i) of mitochondria in the sperematogenic cells of mouse testes 3-24 h after low dose radiation with 0.025-0.200 Gy X-rays, and illuminate the effects of mitochondrion structure and relative biological function on apoptosis. Methods: The ultrastructure changes of mitochondria in the spermatogenic cells were observed with transmission electron microscope; the ATPase activity was measured with protein enzymic method; [Ca 2+ ]i was measured indirectly by flow cytometry with Fluo-3 probes. Results: The mitochondria swelled and vacuolizated, and their cristae were broken in the spermatogonia and spermatocytes 12 h after irradiation, and their nuclei were karyopyknosis, the acrosomal vesicle structure was ambiguity, the membrane structure was unclear, and the mitochondria in spermatids were vacuolization. The activities of Na + -K + -ATPase in mouse testis tissue 12 h after irradiated with 0.025-0.200 Gy decreased compared with those with 0 Gy, the Na + -K + -ATPase activities of the cells irradiated with 0.05-0.200 Gy decreased significantly compared with those with 0 Gy (P 2+ -ATPase of the cells irradiated with 0.025-0.200 Gy decreased significantly compared with those with 0 Gy (P 2+ ]i in mouse testis spermatogenic cells had similar dose-response relationship, [Ca 2+ ]i after irradiated with 0.075 Gy decreased compared with those with 0 Gy (P + -K + -ATPase in mouse testis tissues decreased obviously compared with those at 0 h (P 2+ -ATPase in mouse testis tissues increased slightly at 3 h, then decreased at 6-24 h compared with those at 0 h (P 2+ ]i in mouse testis spermatogenic cells had similar time course-response relationship, [Ca 2+ ]i at 12 h decreased significantly compared with at 0 h (P 2+ ]i induced by low dose radiation. (authors)

  10. Quantitative measurement of membrane Na{sup +}-K{sup +} ATPase activity using thallium-201: comparison with rubidium-86

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae Tae; Shon, Sang Kyun; Lee, Kyu Bo [School of Medicine, Kyungpook National Univ., Taegu (Korea, Republic of); Lee, In Kyu [School of Medicine, Kyemyung Univ., Taegu (Korea, Republic of)

    1998-06-01

    Na{sup +}-K{sup +} ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristics of Tl-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na{sup +}-K{sup +} ATPase activity using Tl-201 and compare with that using Rb-86. Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na{sup +}-K{sup +} ATPase activity. Na{sup +}-K{sup +} ATPase activity was measured as a percentage decrease in cellular uptake of Tl-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Na{sup +}-K{sup +} ATPase activity measured with Tl-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50-70% or 28% increase from baseline activity, respectively. These results suggests that Tl-201 could replace Rb-86 in measurement of ouabain sensitive Na{sup +}-K{sup +} ATPase activity in vitro. High level of glucose concentration decreased cellular Na{sup +}-K{sup +} ATPase activity, but insulin or PMA increased it.

  11. 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.

  12. Nucleotide binding to Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  13. Monoclonal antibody localization of Na sup + -K sup + -ATPase in the exocrine pancreas and parotid of the dog

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Z.D.J.; Caplan, M.J.; Forbush, B. III; Jamieson, J.D. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1987-08-01

    A monoclonal antibody specific to the {beta}-subunit of the canine {sup 125}I-labeled-Na{sup +}-K{sup +}-ATPase has been characterized and used to directly localize the enzyme in thin frozen sections of dog pancreas and parotid. The antibody, 7-2M, recognizes only the {beta}-subunit of the sodium pump as determined by immunoprecipitation and immunoblot and is not directed against an oligosaccharide determinant. 7-2M immunolocalizes to the same cellular and subcellular domains of renal tubular cells as do other, previously characterized, antibodies directed to the {alpha}-subunit of the sodium pump. In the pancreas the preponderance of the Na{sup +}-K{sup +}-ATPase is found on the basolateral membranes of centroacinar and intralobular duct cells. Interlobular duct cells also express a large component of basolaterally located enzyme, although comparatively little pump is seen on acinar cells. In the parotid a large amount of Na{sup +}-K{sup +}-ATPase is seen on the striated cut cells, with high levels also noted on cells of the intercalated ducts and serous demilunes. Again the acinar cells show comparatively low levels of Na{sup +}-K{sup +}-ATPase. In no instance is Na{sup +}-K{sup +}-ATPase found on the apical membranes of pancreas or parotid cells. These data suggest that Na{sup +}-K{sup +}-ATPase, located on the basolateral plasmalemma of duct-derived cells, may be involved in water and electrolyte secretion from the pancreas and parotid.

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

    Science.gov (United States)

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

    1998-01-01

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

  15. Pancreatic Enzymes

    Science.gov (United States)

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  16. Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

    International Nuclear Information System (INIS)

    Haber, R.S.; Loeb, J.N.

    1988-01-01

    The administration of thyroid hormone is known to result in an induction of the Na + -K + -adenosinetriphosphatase (Na + -K + -ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na + -K + -ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3',5-triiodo-L-thyronine (T 3 ) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na + -K + -ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K I s) for ouabain of ∼10 -7 and 10 -4 M, respectively. Measurement of the specific binding of [ 3 H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K d ) of slightly less than 10 -7 M, whose maximal binding capacity was increased by T 3 treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T 3 treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na + -K + -ATPase by ouabain-dependent phosphorylation from [ 32 P]orthophosphate confirmed that T 3 treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na + -K + -ATPase are subject to selective hormonal induction

  17. 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

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

    Science.gov (United States)

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

    2016-10-01

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

  19. Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

    Science.gov (United States)

    Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

    2002-05-01

    White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

  20. Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

    Science.gov (United States)

    Onken, Horst; Patel, Malay; Javoroncov, Margarita; Izeirovski, Sejmir; Moffett, Stacia B; Moffett, David F

    2009-03-01

    Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

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

    Science.gov (United States)

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

    2015-12-01

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

  2. 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.

  3. Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney

    International Nuclear Information System (INIS)

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A.

    1988-01-01

    A partially purified, membrane-bound Na + -K + -ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H 2 O 2 for either 15 or 30 min at 37 degree C. The activity of ouabain-sensitive Na + -K + -ATPase was reduced proportionally to the concentration of H 2 O 2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na + -K + -ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na + K + -ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degree C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na + -K + -ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney

  4. 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.

  5. Characterization of the anion sensitive ATPase in intact vacuoles of Kalanchoe diagremontiana

    Energy Technology Data Exchange (ETDEWEB)

    Kobza, J.; Uribe, E.G.

    1986-04-01

    A method for the isolation of intact vacuoles from K. daigremontiana was developed which produced high yields of relatively pure vacuoles as determined by marker enzyme contamination. Upon isolation, the vacuoles were stabilized by the inclusion of 5% (w/v) ficoll. Enzyme activity was insensitive to vanadate and azide but was strongly inhibited by DCCD. Enzyme activity was strictly dependent on the inclusion of Mg/sup 2 +/ and was stimulated by anions as depicted by the series, NO/sub 3//sup -/ < Br/sup -/ < SO/sub 4//sup -/ < HCO/sub 3//sup -/ < Cl/sup -/. It was found that in intact vacuoles the ATPase activity was stimulated by phosphate to a level equivalent to that found with the chloride. The enzyme exhibited Michaelis-Menten kinetics with a Km for Mg-ATP complex of 0.51 mM.

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

    Science.gov (United States)

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

    2007-01-01

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

  7. 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)

  8. In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

    Directory of Open Access Journals (Sweden)

    L.M. Zanatta

    2001-10-01

    Full Text Available The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10% in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27% in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group. When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80% occurring at 0.5 mM. We suggest that a imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

  9. Characterization of a bacteriophage T4 mutant lacking DNA-dependent ATPase

    International Nuclear Information System (INIS)

    Behme, M.T.; Ebisuzaki, K.

    1975-01-01

    A DNA-dependent ATPase has previously been purified from bacteriophage T4-infected Escherichia coli. A mutant phage strain lacking this enzyme has been isolated and characterized. Although the mutant strain produced no detectable DNA-dependent ATPase, growth properties were not affected. Burst sizes were similar for the mutant phage and T4D in polAl, recB, recC, uvrA, uvrB, uvrC, and various DNA-negative E. coli. UV sensitivity and genetic recombination were normal in a variety of E. coli hosts. Mapping data indicate that the genetic locus controlling the mutant occurs near gene 56. The nonessential nature of this gene is discussed

  10. Investigations into the binding of 125I-calmodulin to CA++ transport ATPase of human erythrocytes

    International Nuclear Information System (INIS)

    Sterk, V.

    1983-01-01

    The study described was carried out in order to investigate the binding of 125 I-calmodulin to Ca ++ transport ATPase using different Ca ++ concentrations and temperatures. The data obtained from these experiments were subsequently analysed in such as a way as to yield meaningful information relating to the mechanisms underlying the attachment of calmodulin to Ca ++ transport ATPase, the % proportion of membrane protein that was attributable to the enzyme as well as the number of calmodulin receptor sites on the individual erythrocytes, etc. Comparisons with data from the relevant literature permitted conclusions to be drawn concerning the mode of Ca ++ transport at the level of the erythrocytes. A new methodology and processing technique had to be developed prior to the beginning of the experiments. (orig./MG) [de

  11. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.

    Science.gov (United States)

    Costa, Sara R; Marek, Magdalena; Axelsen, Kristian B; Theorin, Lisa; Pomorski, Thomas G; López-Marqués, Rosa L

    2016-06-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  12. On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

    International Nuclear Information System (INIS)

    Du, Z.; Boyer, P.D.

    1990-01-01

    Washed chloroplast thylakoid membranes upon exposure to [ 3 H]ADP retain in tightly bound [ 3 H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg 2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [ 3 H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg 2+ - and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22 degree C and of about 15 s at 37 degree C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg 2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [ 3 H]ADP parallels the onset of ATPase activity, although some [ 3 H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [ 3 H]ADP being at a catalytic site and being replaced as this Mg 2+ - and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P i binding site of the enzyme-ADP-Mg 2+ complex to give a form more readily activated by ATP binding at an alternative site

  13. 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.

  14. 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

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

    International Nuclear Information System (INIS)

    Milgrom, Elena M.; Milgrom, Yakov M.

    2012-01-01

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

  16. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  17. 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.

  18. Ionic Liquid-Promoted Synthesis of Novel Chromone-Pyrimidine Coupled Derivatives, Antimicrobial Analysis, Enzyme Assay, Docking Study and Toxicity Study

    Directory of Open Access Journals (Sweden)

    Shailee V. Tiwari

    2018-02-01

    Full Text Available Herein, we report an environmentally friendly, rapid, and convenient ionic liquid ([Et3NH][HSO4]-promoted facile synthesis of ethyl 4-(6-substituted-4-oxo-4H-chromen-3-yl-6-methyl-2-thioxo/oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 4(a–f and 4-(6-substituted-4-oxo-4H-chromen-3-yl-6-methyl-2-thioxo/oxo-1,2,3,4-tetrahydropyrimidine-5- carbohydrazide derivatives 6(a–f. All the synthesized derivatives 4(a–f and 6(a–f were evaluated for their in vitro antifungal and antibacterial activity, by method recommended by National Committee for Clinical Laboratory Standards (NCCLS. The compound 6c bearing a fluoro group on the chromone ring and oxygen and a hydrazino group (–NHNH2 on the pyrimidine ring, was found to be the most potent antibacterial compound amongst the synthesized derivatives. The compound 6f bearing a methoxy group (–OCH3 on the chromone ring and sulphur group on the pyrimidine ring, was found to exhibit equipotent antifungal activity when compared with the standard drug miconazole. A d-alanine-d-alanine ligase (DdlB enzyme assay study and an ergosterol extraction and quantitation assay study were performed to predict the mode of action of the synthesized compounds. A molecular docking study was performed to predict the binding interactions with receptors and mode of action of the synthesized derivatives. Further, analysis of the ADMET parameters for the synthesized compounds has shown that these compounds have good oral drug-like properties and can be developed as oral drug candidates. To establish the antimicrobial selectivity and safety, the most active compounds 6c and 6f were further tested for cytotoxicity against the human cancer cell line HeLa and were found to be non-cytotoxic in nature. An in vivo acute oral toxicity study was also performed for the most active compounds 6c and 6f and the results indicated that the compounds are non-toxic in nature.

  19. Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state.

    Science.gov (United States)

    Kanai, Ryuta; Ogawa, Haruo; Vilsen, Bente; Cornelius, Flemming; Toyoshima, Chikashi

    2013-10-10

    Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.

  20. 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

  1. Overproduction of PIB-Type ATPases

    DEFF Research Database (Denmark)

    Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo

    2016-01-01

    Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purifi...... and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals....

  2. Effects of solubilization on the inhibition of the p-type ATPase from maize roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

    Science.gov (United States)

    Brauer, D K; Gurriel, M; Tu, S I

    1992-12-01

    The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.

  3. Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos.

    Science.gov (United States)

    Zivkovic, Danica; Créton, Robbert; Dohmen, René

    1991-08-01

    During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca 2+ -stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm 2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca 2+ /Mg 2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca 2+ -stimulated ATPase. The ionic currents and Ca 2+ -stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca 2+ -stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca 2+ -stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.

  4. 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...

  5. 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.

  6. Effect of green laser light on diabetes mellitus changed ATPase activity in erythrocytes

    International Nuclear Information System (INIS)

    Kassak, P.; Sikurova, L.

    2006-01-01

    Changes in the membrane bound enzyme activity may report about changes of processes and properties related to the cytoplasmic membrane of cells. Activity of the Na + /K + -ATPase has become objective of our investigation as o tool to evaluate changes of diabetic membranes in comparison to normal membranes of human erythrocytes after laser irradiation with Nd:YAG laser (532 nm) in fluence range 9.5-63.3 J · cm -2 . Energies of irradiation 3-20 joules and output power of the laser 30 mW classify this experiment as low-level laser therapy. Bio-stimulation of the enzyme, its activity as well as type-2 diabetes caused disorganisation and alternation of biological membrane and enzyme properties are discussed. (Authors)

  7. Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

    International Nuclear Information System (INIS)

    Steinberg, T.H.; Silverstein, S.C.

    1987-01-01

    Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86 Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86 Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86 Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86 Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86 Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86 Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-

  8. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

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

  9. 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.

  10. 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.

  11. Effect of ionizing radiation on catalytic properties of Ca2+-ATP-ase from sarcoplasmic reticulum of skeletal muscle

    International Nuclear Information System (INIS)

    Bagel', I.M.; Shafranovskaya, E.V.; Gorokh, G.A.; Markova, A.G.

    1999-01-01

    It was studied kinetic and thermodynamic characteristics of Ca 2+ -ATP-ase of rat skeletal muscle (membranes of sarcoplasmic reticulum) after irradiation in doses 0,5, 4,0 and 8,0 Gy. It was shown that external gamma-irradiation at different doses changed kinetic and thermodynamic characteristics of the enzyme of sarcoplasmic reticulum membranes of skeletal muscle. These alterations probably correlate with disbalance of hormonal regulation of intracellular calcium metabolism and changes in membrane structure and functions

  12. Role of the Na+/K+-ATPase ion pump in male reproduction and embryo development.

    Science.gov (United States)

    Câmara, D R; Kastelic, J P; Thundathil, J C

    2017-08-01

    Na + /K + -ATPase was one of the first ion pumps studied because of its importance in maintaining osmotic and ionic balances between intracellular and extracellular environments, through the exchange of three Na + ions out and two K + ions into a cell. This enzyme, which comprises two main subunits (α and β), with or without an auxiliary polypeptide (γ), can have specific biochemical properties depending on the expression of associated isoforms (α1β1 and/or α2β1) in the cell. In addition to the importance of Na + /K + -ATPase in ensuring the function of many tissues (e.g. brain, heart and kidney), in the reproductive tract this protein is essential for embryo development because of its roles in blastocoel formation and embryo hatching. In the context of male reproduction, the discovery of a very specific subunit (α4), apparently restricted to male germ cells, only expressed after puberty and able to influence sperm function (e.g. motility and capacitation), opened a remarkable field for further investigations regarding sperm biology. Therefore, the present review focuses on the importance of Na + /K + -ATPase on male reproduction and embryo development.

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

    International Nuclear Information System (INIS)

    Kim, M.S.; Akera, T.

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, M.S.; Akera, T.

    1987-02-01

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

  15. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Directory of Open Access Journals (Sweden)

    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  16. High Level Soluble Expression and ATPase Characterization of Human Heat Shock Protein GRP78.

    Science.gov (United States)

    Wu, Shuang; Zhang, Hongpeng; Luo, Miao; Chen, Ke; Yang, Wei; Bai, Lei; Huang, Ailong; Wang, Deqiang

    2017-02-01

    Human GRP78 has been shown to promote cancer progression and is regarded as a novel target for anticancer drugs. However, generation of recombinant full-length GRP78 remains challenging. This report demonstrates that E. coli autoinduction is an excellent method for the preparation of active recombinant GRP78 protein. The final yield was approximately 50 mg/liter of autoinduction culture. Gel-filtration experiments confirmed that the chaperone is a monomer. The purified human GRP78 catalyzed the conversion of ATP to ADP without requiring metal ions as cofactors. Three mutants, T38A, T229A, and S300A, exhibited much lower activity than wild-type GRP78, indicating that the active sites of the ATPase are located at the negatively charged cavity. Three mutants in the negatively charged cavity region dramatically reduced GRP78 activity, further confirming the region as the site of ATPase activity.

  17. 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.

  18. Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland.

    Science.gov (United States)

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

    2011-02-01

    Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC(50)=0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F(0), either in the form of free enzyme or of enzyme-substrate complex (Ki=K'i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg(2+) cofactor, may bind strongly to F(1) subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na(+), K (+))- and Mg ( 2+ )-ATPase activities of adult rat hypothalamus and cerebellum.

    Science.gov (United States)

    Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

    2007-03-01

    Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na(+), K(+))- and Mg(2+)-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (-23%, p activities (-26%, p activities: AChE (-17%, p activity was found unaltered in both the hyper- and the hypothyroid brain regions. neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na(+), K(+)-ATPase activities. The decreased (by the THs) Na(+), K(+)-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.

  20. 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.

  1. Ion pump as molecular ratchet and effects of noise: electric activation of cation pumping by Na,K-ATPase

    Science.gov (United States)

    Tsong, T. Y.; Xie, T. D.

    2002-08-01

    Na,K-ATPase is a universal ion pump of the biological cell. Under physiological conditions, it uses the γ-phosphorus bond energy of ATP during hydrolysis to pump 2 K+ inward and 3 Na+ outward; both being uphill transports. The experiment presented here demonstrates that the protein transporter can also use electric energy to fuel its pump activity. A theory of electroconformational coupling (TEC) is described and an experiment performed to verify several predictions of the model. Analysis based on the TEC model suggests that Na,K-ATPase is a Brownian ratchet. The enzyme harvests energy from the applied field by means of the field-induced conformational oscillation or fluctuation. However, high efficiency of energy transduction can only be achieved with an electric field of certain intensities, frequencies and waveforms. This property of the enzyme allows us to define an electric signal and differentiate it from electric noise on the basis of the analysis by the TEC model. Data show that electric noise alone does not induce pump activity. However, an appropriate power level of noise imposed on a signal can enhance the pump efficiency. The effect of noise on the signal transduction of Na,K-ATPase is reminiscent of the stochastic resonance phenomenon reported in other biological systems [3, 35]. The TEC model embodies many common features of enzymes and biological motors. It is potentially energy-efficient, much more so than models based on the ion-rectification mechanism.

  2. 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

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

  4. The role of Na+/K+-ATPase during chick skeletal myogenesis.

    Directory of Open Access Journals (Sweden)

    Taissa Neustadt Oliveira

    Full Text Available The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na+/K+-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na+/K+-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na+/K+-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na+/K+-ATPase.

  5. The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.

    Science.gov (United States)

    Camire, Eric J; Grossman, John D; Thole, Grace J; Fleischman, Nicholas M; Perlstein, Deborah L

    2015-09-25

    Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 μM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells

    International Nuclear Information System (INIS)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J.

    1988-01-01

    Dopamine, generated locally from L-dopa, inhibits Na + -K + -ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na + -K + -ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (Qo 2 ) and 86 Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive Qo 2 or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive Qo 2 in a concentration-dependent manner, with half-maximal inhibition (K 0.5 ) of 5 x 10 -7 M and a maximal inhibition of 14.1 ± 1.5% at 10 -4 M. L-Dopa also blunted the nystatin-stimulated Qo 2 in a concentration-dependent manner, indicating the L-dopa directly inhibits Na + -K + -ATPase activity and not sodium entry. Ouabain-sensitive 86 Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive Qo 2 and 86 Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive Qo 2 at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na + -K + -ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner

  7. Gill Na+,K+-ATPase of Atlantic salmon smolts in freshwater is not a predictor of long-term growth in seawater

    Science.gov (United States)

    Zydlewski, Gayle B.; Zydlewski, Joseph D.

    2012-01-01

    Gill Na+,K+-ATPase activity is a widely used measure of osmoregulatory preparedness in salmonid smolts. The degree to which this measure may predict long term performance is uncertain. In order to assess the relationship of this enzyme to long term growth and ion homeostasis, a cohort of Atlantic salmon hatchery smolts was used in a controlled environment with no salinity perturbations. In May 2006, gill Na+,K+-ATPase activity from 940 individually PIT tagged, Penobscot River smolts (USFWS, Green Lake National Fish Hatchery, Maine, United States) was measured immediately prior to isothermal transfer from freshwater to 32 ppt seawater. From the observed range of activities, individuals were classified as having “low”, “middle”, or “high” enzyme activity levels. Individual size (fork length and mass) was recorded on days 0, 1, 3, and 14 and monthly for four months. Growth rates over four time periods were calculated for individual fish maintained until the end of the experiment. Gill Na+,K+-ATPase activities were also measured from a subset of sampled fish. All groups effectively osmoregulated as evidenced by minor perturbations in plasma osmolyte levels. Apart from initial weight loss on transfer, fish grew throughout the experiment, however, there were no differences (fish size, growth rate, and gill Na+,K+-ATPase activity in seawater) among groups with initially different gill Na+,K+-ATPase activities (prior to seawater entry). While gill Na+,K+-ATPase activity may be predictive of performance during the acute phase of acclimation (first few days), typical variation in this enzyme, expressed in freshwater at the peak of smolting, does not appear to be predictive of long-term growth in seawater.

  8. Kidney in potassium depletion. I. Na/sup +/-K/sup +/-ATPase activity and (/sup 3/H)ouabain binding in MCT

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, M.; Katz, A.I.

    1987-03-01

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

  9. 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...

  10. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

    Science.gov (United States)

    Koues, Olivia I; Mehta, Ninad T; Truax, Agnieszka D; Dudley, R Kyle; Brooks, Jeanne K; Greer, Susanna F

    2010-02-04

    Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription. Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  11. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

    Directory of Open Access Journals (Sweden)

    Koues Olivia I

    2010-02-01

    Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  12. OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo

    DEFF Research Database (Denmark)

    Iglesias-Gato, Diego; Chuan, Yin-Choy; Jiang, Ning

    2015-01-01

    BackgroundUbiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored.MethodsRNAi screening was us...

  13. Activity of Ca(2+,Mg(2+-ATPase of sarcoplasmic reticulum and contraction strength of the frog skeletal muscles under the effect of organophosphorus insecticides

    Directory of Open Access Journals (Sweden)

    D. M. Nozdrenko

    2015-08-01

    Full Text Available The results of an experimental study of organo­phosphorus insecticides, including pirimiphosmethyl, diazinon and chlorpyrifos caused a decline of the contraction properties in m. tibialis anterior fiber bundles of Rana temporaria, as well as sarcoplasmic reticulum Ca2+,Mg2+-ATPase enzymatic activity reduction are outlined in this paper. Concentration-dependent strengths response diminishing in isolated skeletal muscle fiber bundles as a result of non-cholinergic influence of organophosphorus insecticides were found. A decrease of Ca2+,Mg2+-ATPase enzymatic activity in sarcoplasmic reticulum was observed after administration of each insecticide. The most significant inhibition of this enzyme was observed when using chlorpyrifos.

  14. [ACTIVITY OF Ca2+,Mg(2+)-ATPase OF SARCOPLASMIC RETICULUM AND CONTRACTION STRENGTH OF THE FROG SKELETAL MUSCLES UNDER THE EFFECT OF ORGANOPHOSPHORUS INSECTICIDES].

    Science.gov (United States)

    Nozdrenko, D M; Korchinska, L V; Soroca, V M

    2015-01-01

    The results of an experimental study of organophosphorus insecticides, including pirimiphosmethyl, diazinon and chlorpyrifos caused a decline of the contraction properties in m. tibialis anterior fiber bundles of Rana temporaria, as well as sarcoplasmic reticulum Ca2+, Mg(2+)-ATPase enzymatic activity reduction are outlined in this paper. Concentration-dependent strengths response diminishing in isolated skeletal muscle fiber bundles as a result of non-cholinergic influence of organophosphorus insecticides were found. A decrease of Ca2+, Mg(2+)-ATPase enzymatic activity in sarcoplasmic reticulum was observed after administration of each insecticide. The most significant inhibition of this enzyme was observed when using chlorpyrifos.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  16. Effect of thyroid status on the development of the different molecular forms of Na+,K+-ATPase in rat brain.

    Science.gov (United States)

    Atterwill, C K; Reid, J; Athayde, C M

    1985-05-01

    The effect of thyroid status on the postnatal development of the two molecular forms of Na+,K+-ATPase, distinguished kinetically on the basis of their ouabain sensitivity, was examined in rat brain. Hypothyroidism induced by PTU from day 1 postnatally significantly reduced the Na+,K+-ATPase activity in cerebellum (22-30 days) but not forebrain, whereas hyperthyroidism (T4 treatment from day 1) had no effect. The hypothyroidism-induced reduction in cerebellum was reflected by a 20-45% reduction in the activity of the alpha(+) form of Na+,K+-ATPase (high ouabain affinity) against control brains compared to a 60-70% reduction in the activity of the alpha form (low ouabain affinity). These results show that neonatally induced hypothyroidism leads to a selectively greater impairment of the ontogenesis of the activity of cerebellar alpha form of Na+,K+-ATPase. This may possibly reflect a retarded development of a selective cerebellar cell population containing predominantly the alpha form of the enzyme.

  17. Na, K-ATPase as signaling transducer

    OpenAIRE

    Li, Juan

    2007-01-01

    It is now generally agreed that Na,K-ATPase (NKA), in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, is a signal transducer. Our group has identified a novel signaling pathway where NKA interact with IP3R to form a signaling microdomain. Ouabain, a specific ligand of NKA, activates this pathway, triggers slow Ca2+ oscillations and activates NF-κB. In current study, the molecular mechanisms and some important downstream effects of NK...

  18. 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...

  19. Kinase-Mediated Regulation of P4-ATPases

    DEFF Research Database (Denmark)

    Frøsig, Merethe Mørch

    designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps...

  20. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  1. 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.

  2. Dual function of Swc5 in SWR remodeling ATPase activation and histone H2A eviction.

    Science.gov (United States)

    Sun, Lu; Luk, Ed

    2017-09-29

    The chromatin remodeler SWR deposits histone H2A.Z at promoters and other regulatory sites via an ATP-driven histone exchange reaction that replaces nucleosomal H2A with H2A.Z. Simultaneous binding of SWR to both H2A nucleosome and free H2A.Z induces SWR ATPase activity and engages the histone exchange mechanism. Swc5 is a conserved subunit of the 14-polypeptide SWR complex that is required for the histone exchange reaction, but its molecular role is unknown. We found that Swc5, although not required for substrate binding, is required for SWR ATPase stimulation, suggesting that Swc5 is required to couple substrate recognition to ATPase activation. A biochemical complementation assay was developed to show that a unique, conserved domain at the C-terminus of Swc5, called Bucentaur (BCNT), is essential for the histone exchange activity of SWR, whereas an acidic region at the N-terminus is required for optimal SWR function. In vitro studies showed the acidic N-terminus of Swc5 preferentially binds to the H2A-H2B dimer and exhibits histone chaperone activity. We propose that an auxiliary function of Swc5 in SWR is to assist H2A ejection as H2A.Z is inserted into the nucleosome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Effect of lithium and of other drugs used in the treatment of manic illness on the cation-transporting properties of Na+,K+-ATPase in mouse brain synaptosomes

    Energy Technology Data Exchange (ETDEWEB)

    Wood, A J; Elphick, M; Grahame-Smith, D G

    1989-04-01

    We have developed and used a novel technique to investigate the effects of lithium and other psychotropic drugs on the cation-transporting properties of the sodium- and potassium-activated ATPase enzyme (Na+,K+-ATPase) in intact synaptosomes. Rubidium-86 uptake into intact synaptosomes is an active process and is inhibited by approximately 75% in the presence of the Na+,K+-ATPase inhibitor acetylstrophanthidin. In vitro addition of lithium to synaptosomes prepared from untreated mice causes a progressive inhibition of acetylstrophanthidin-sensitive /sup 86/Rb uptake, but only at concentrations higher than the clinical therapeutic range. However, pretreatment of mice for 14 days in vivo with lithium, carbamazepine, and haloperidol, but not phenytoin, causes a significant stimulation of /sup 86/Rb uptake into synaptosomes via Na+,K+-ATPase.

  4. 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.

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

  7. Inhibition and labeling of the plant plasma membrane H+-ATPase with N-ethylmaleimide

    International Nuclear Information System (INIS)

    Katz, D.B.; Sussman, M.R.

    1987-01-01

    H + -ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated with N-[2- 3 H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyaerylamide gel electrophoresis, prominent radioactive bands appear at M/sub r/ = 100,000 and several other positions. However, only radioactivity in the M/sub r/ = 100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the M/sub r/ = 100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H + -ATPase. When tryptic peptides are produced from N-[2- 3 H]ethylmaleimide labeled M/sub r/ = 100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2- 3 H]ethylmaleimide incorporation is reduced in the presence of MgADP

  8. 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.

  9. A novel fluorescence imaging approach to monitor salt stress-induced modulation of ouabain-sensitive ATPase activity in sunflower seedling roots.

    Science.gov (United States)

    Mukherjee, Soumya; Bhatla, Satish Chander

    2014-04-01

    Seedlings exposed to salt stress are expected to show modulation of intracellular accumulation of sodium ions through a variety of mechanisms. Using a new methodology, this work demonstrates ouabain (OU)-sensitive ATPase activity in the roots of sunflower seedlings subjected to salt stress (120 mM NaCl). 9-Anthroylouabain (a derivative of ouabain known to inhibit Na(+), K(+) -ATPase activity in animal systems, EC 3.6.3.9) has been used as a probe to analyze OU-sensitive ATPase activity in sunflower (Helianthus annuus) seedling roots by spectrofluorometric estimation and localization of its spatial distribution using confocal laser scanning microscopy. Salt stress for 48 h leads to a significant induction of OU-sensitive ATPase activity in the meristematic region of the seedling roots. Calcium ions (10 mM) significantly inhibit enzyme activity and a parallel accumulation of sodium ions in the cytosol of the columella cells, epidermis and in the cells of the meristematic region of the roots is evident. As a rapid response to NaCl stress, the activity of OU-sensitive ATPase gets localized in the nuclear membrane of root protoplasts and it gets inhibited after treatment with calcium ions. Nuclear membrane localization of the OU-sensitive ATPase activity highlights a possible mechanism to efflux sodium ions from the nucleus. Thus, a correlation between OU-sensitive ATPase activity, its modulation by calcium ions and accumulation of sodium ions in various regions of the seedling roots, has been demonstrated using a novel approach in a plant system. © 2013 Scandinavian Plant Physiology Society.

  10. Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

    International Nuclear Information System (INIS)

    Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

    1987-01-01

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

  11. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  12. Factors Influencing the Increase in Na-K-ATPase in Compensatory Renal Hypertrophy

    Science.gov (United States)

    Epstein, Franklin H.; Charney, Alan N.; Silva, Patricio

    1978-01-01

    An increase in Na-K-ATPase in kidney homogenates usually accompanies compensatory renal hypertrophy. While it may be evident in both the cortex and medulla of the kidney, it is most marked in the outer medulla and may be present only in that region. The increase in enzyme activity does not depend on an intact adrenal cortex and can be elicited in the absence of adrenal glucocorticoids. It is not seen in the form of renal hypertrophy produced by potassium depletion, in which the transport of sodium and potassium by the kidney is not increased. When present in compensatory renal growth, the enzyme change is correlated with an increase in the reabsorption of sodium, or the excretion of potassium, or both, per unit of renal tissue. It proceeds in the presence of either, but not in the absence of both. PMID:216164

  13. Combination of DFP and Taurine Counteracts the Aluminum-Induced Alterations in Oxidative Stress and ATPase in Cortex and Blood of Rats.

    Science.gov (United States)

    Feng, Tong; Liu, Ping; Zhang, Zhen; Hu, Jinyu; Kong, Zhengqiao

    2016-11-01

    The study investigated the combined effect of 1,2-dimethyl-3-hydroxypyrid-4-one (DFP) and taurine on aluminum (Al) toxicity in cortex and blood of rats. The control group received 1 ml/kg/day saline solution for 8 weeks. Other animals were exposed to Al at a dose of 281.40 mg/kg/day orally for 4 weeks. Then, they were administered with 1 ml/kg/day saline solution, 400 mg/(kg·day) taurine, 13.82 mg/(kg·day) DFP, 27.44 mg/(kg·day) DFP, 400 mg/(kg·day) taurine +13.82 mg/(kg·day) DFP, and 400 mg/(kg·day) taurine +27.44 mg/(kg·day) DFP for 4 weeks. The changes in markers of oxidative stress, activities of antioxidant enzymes, and triphosphatase (ATPase) in the cortex and blood were determined. Administration of Al led to significant increase in the malondialdehyde (MDA) level and decrease in the activities of antioxidant enzymes, Na + K + -ATPase, Mg 2+ -ATPase, and Ca 2+ -ATPase in the cortex and blood, compared with the control group. DFP was observed to reverse alteration of these parameters except for Ca 2+ -ATPase activity. Treatment with taurine caused significant increase of GSH-Px activity and decrease of the MDA level in the cortex and serum and rise of Na + K + -ATPase in the blood. Effects of DFP combined with taurine were investigated and found to provide a more significant benefit than either drug alone. Combined intake of taurine and DFP could achieve an optimum effect of therapy for Al exposure.

  14. Oxidative stress and damage to erythrocytes in patients with chronic obstructive pulmonary disease--changes in ATPase and acetylcholinesterase activity.

    Science.gov (United States)

    Bukowska, Bożena; Sicińska, Paulina; Pająk, Aneta; Koceva-Chyla, Aneta; Pietras, Tadeusz; Pszczółkowska, Anna; Górski, Paweł; Koter-Michalak, Maria

    2015-12-01

    The study indicates, for the first time, the changes in both ATPase and AChE activities in the membrane of red blood cells of patients diagnosed with COPD. Chronic obstructive pulmonary disease (COPD) is one of the most common and severe lung disorders. We examined the impact of COPD on redox balance and properties of the membrane of red blood cells. The study involved 30 patients with COPD and 18 healthy subjects. An increase in lipid peroxidation products and a decrease in the content of -SH groups in the membrane of red blood cells in patients with COPD were observed. Moreover, an increase in the activity of glutathione peroxidase and a decrease in superoxide dismutase, but not in catalase activity, were found as well. Significant changes in activities of erythrocyte membrane enzymes in COPD patients were also evident demonstrated by a considerably lowered ATPase activity and elevated AChE activity. Changes in the structure and function of red blood cells observed in COPD patients, together with changes in the activity of the key membrane enzymes (ATPases and AChE), can result from the imbalance of redox status of these cells due to extensive oxidative stress induced by COPD disease.

  15. Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

    Science.gov (United States)

    Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan

    2014-12-01

    Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  17. 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...

  18. 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,...

  19. Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression.

    Science.gov (United States)

    Khatua, Tarak N; Borkar, Roshan M; Mohammed, Soheb A; Dinda, Amit K; Srinivas, R; Banerjee, Sanjay K

    2017-01-01

    Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg -1 day -1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na + /K + -ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na + /K + -ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na + /K + -ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na + /K + -ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na + /K + -ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy.

  20. Depression of membrane-bound Na sup + -K sup + -ATPase activity induced by free radicals and by ischemia of kidney

    Energy Technology Data Exchange (ETDEWEB)

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A. (Univ. of Ottawa, Ontario (Canada))

    1988-02-01

    A partially purified, membrane-bound Na{sup +}-K{sup +}-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H{sub 2}O{sub 2} for either 15 or 30 min at 37{degree}C. The activity of ouabain-sensitive Na{sup +}-K{sup +}-ATPase was reduced proportionally to the concentration of H{sub 2}O{sub 2} and the duration of incubation. There were decreases in SH contents and turnover rates of the Na{sup +}-K{sup +}-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na{sup +}K{sup +}-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4{degree}C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na{sup +}-K{sup +}-ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney.

  1. Gill Na{sup +}, K{sup +}-ATPase activity in largemouth bass (Micropterus salmoides) inhabiting reservoirs contaminated with mercury

    Energy Technology Data Exchange (ETDEWEB)

    Brundage, S.; Jagoe, C.H.; Shaw-Allen, P. [Univ. of Georgia, Aiken, SC (United States). Savannah River Ecology Lab.

    1995-12-31

    Active transport of Na{sup +} and K{sup +} for osmoregulation in fish involves gill Na{sup +}, K{sup +}-ATPase, a membrane-bound enzyme powered by hydrolysis of ATP. Na{sup +}, K{sup +}-ATPase is inhibited by many dissolved metals including Al, Cd, Cu and Hg, resulting in ionoregulatory dysfunction. However, dissolved Hg concentrations are quite low in most aquatic systems, and dietary sources are the most important contributors to Hg burdens in fish. One recent study demonstrated relationships between muscle Hg concentration and gill Na{sup +}, K{sup +}-ATPase in a marine fish, suggesting that Hg accumulated via diet can affect osmoregulation. The authors tested for such a relationship in several age-classes of a freshwater fish (Micropterus salmoides) collected from three reservoirs. Fish from Par Pond and L Lake, on the USDOE Savannah River Site in South Carolina had relatively high Hg content: for Par Pond, muscle and liver ranged from 1.58--12.01 and 1.46--23.22 {micro}g Hg/g dry mass, respectively, and for L Lake muscle and liver ranged from 3.11--5.16 and 1.28--12.59 {micro}g Hg/g dry mass, respectively. Bass from an offsite location, Thurmond Lake, had significantly (P <0.05 by Kruskal-Wallis test) less Hg (muscle and liver range 0.61--2.39 and 0.28--2.32 {micro}g Hg/g dry mass, respectively). In all reservoirs, liver Hg varied more among individuals than muscle Hg. Water chemistry was similar in all reservoirs. Fish from the three reservoirs did not differ significantly in gill ATPase activity, and a correlation between tissue Hg and Na{sup +}, K{sup +}-ATPase activity was not evident.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  3. [Comparative investigation of the effect of calix[4]arene C-99 and its analogs on Na+,K(+)-ATPase activity of uterus myocite plasma membrane].

    Science.gov (United States)

    Veklich, T O; Shkrabak, O A; Cherenok, S O; Kal'chenko, V I; Kosterin, S O

    2012-01-01

    The aim of our investigation was to determine structural features of calix[4]arene C-99 which are important for its inhibition properties relative to Na+,K(+)-ATPase of uterus myocite plasma membrane. Therefore we studied the effect of calix[4]arenes C-296, C-297, C-424, C-425, C-426, C-427, which are structurally similar to this inhibitor, on the mentioned enzyme activity. We have shown that calixarenes C-296 and C-297 which have two additional propoxy groups on the lower rim of macrocycle are less effective inhibitors of Na+,K(+)-ATPase relative to calixarene C-99. Calixarenes C-425 and C-427 which have on the upper rim of macrocycle three and four phosponic residues, respectively, also inhibit Na+,K(+)-ATPase activity less effectively as compared to calixarene C-99. Both calixarenes: C-424, which has only two carbonate residues on the upper rim, and C-426, which has on the upper rim ketomethilphosphonate residues instead of hydroxymethilphosphonate residues of calixarene C-99, do not affect Na+,K(+)-ATPase activity. We have made respective conclusions concerning the role of certain chemical groups of calixarene C-99 during its interaction with Na+,K(+)-ATPase.

  4. The effect of different doses of epidermal growth factor on liver ornithine decarboxylase and Na-K ATPase activities in newborn rats.

    Science.gov (United States)

    Bilgihan, A; Turkozkan, N; Isman, F; Kilinc, M; Demirsoy, S

    1998-08-01

    1. Ornithine decarboxylase and Na-K ATPase activities were studied in rat livers that were treated with different doses of epidermal growth factor (EGF). 2. The ornithine decarboxylase activities were studied with spectrophotometry, and results were expressed as micromoles of putrescine per hour per milligram of protein. Na-K ATPase activities were studied on the basis of the principle of measuring the amount of inorganic phosphates released by the hydrolysis of ATP, and the results were expressed as micromoles of inorganic phosphate per hour per milligram of protein. 3. When compared with the controls, although the Na-K ATPase activities were decreased at low doses of EGF, their activities were found to be increased at high doses of EGF. On the other hand, there was a positive correlation between ornithine decarboxylase activities and EGF doses. 4. The results of this study suggest that, whereas the decrease in Na-K ATPase activities at low doses of EGF can be due to the utilization of the enzyme, the increase in Na-K ATPase activities at high doses of EGF can be attributed to its enhanced synthesis.

  5. 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.

  6. 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.

  7. Nicotine promotes cell proliferation via α7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    International Nuclear Information System (INIS)

    Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin

    2007-01-01

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a β 1 - and β 2 -selective antagonist, respectively, suggesting the role of β-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of α7-nicotinic acetylcholine receptor (α7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an α7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and DβH expression as well as adrenaline production. Taken together, through the action on α7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and β-adrenergic activation. These data reveal the contributory role α7-nAChR and β-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer

  8. 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...

  9. Time-dependent inhibition of Na+/K+-ATPase induced by single and simultaneous exposure to lead and cadmium

    Science.gov (United States)

    Vasić, V.; Kojić, D.; Krinulović, K.; Čolović, M.; Vujačić, A.; Stojić, D.

    2007-09-01

    Time-dependent interactions of Na+/K+-ATPase, isolated from rat brain synaptic plasma membranes (SPMs), with Cd2+ and Pb2+ ions in a single exposure and in a mixture were investigated in vitro. The interference of the enzyme with these metal ions was studied as a function of different protein concentrations and exposure time. The aim of the work was to investigate the possibility of selective recognition of Cd2+ and Pb2+ ions in a mixture, on the basis of the different rates of their protein-ligand interactions. Decreasing protein concentration increased the sensitivity of Na+/K+-ATPase toward both metals. The selectivity in protein-ligand interactions was obtained by variation of preincubation time (incubation before starting the enzymatic reaction).

  10. Kinesin-2 KIF3AB exhibits novel ATPase characteristics.

    Science.gov (United States)

    Albracht, Clayton D; Rank, Katherine C; Obrzut, Steven; Rayment, Ivan; Gilbert, Susan P

    2014-10-03

    KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm(-1) s(-1), followed by rate-limiting ADP release at 12.8 s(-1). ATP binding at 7.5 μm(-1) s(-1) was followed by an ATP-promoted isomerization at 84 s(-1) to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s(-1). ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s(-1). The dissociation step showed an apparent affinity for ATP that was very weak (K½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm(-1) s(-1), which is inconsistent with fast ATP binding at 7.5 μm(-1) s(-1) and a Kd ,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K½,ATP at 133 μm. The steady-state ATPase Km ,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. 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.

  12. Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2.

    Directory of Open Access Journals (Sweden)

    Layla El Moussawi

    Full Text Available Epinephrine, a key stress hormone, is known to affect ion transport in the colon. Stress has been associated with alterations in colonic functions leading to changes in water movements manifested as diarrhea or constipation. Colonic water movement is driven by the Na+-gradient created by the Na+/K+-ATPase. Whether epinephrine acts via an effect on the Na+/K+-ATPase hasn't been studied before. The aim of this work was to investigate the effect of epinephrine on the Na+/K+-ATPase and to elucidate the signaling pathway involved using CaCo-2 cells as a model. The activity of the Na+/K+-ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. Epinephrine, added for 20 minutes, decreased the activity of the Na+/K+-ATPase by around 50%. This effect was found to be mediated by α2 adrenergic receptors as it was fully abolished in the presence of yohimbine an α2-blocker, but persisted in presence of other adrenergic antagonists. Furthermore, treatment with Rp-cAMP, a PKA inhibitor, mimicked epinephrine's negative effect and didn't result in any additional inhibition when both were added simultaneously. Treatment with indomethacin, PP2, SB202190, and PD98059, respective inhibitors of COX enzymes, Src, p38MAPK, and ERK completely abrogated the effect of epinephrine. The effect of epinephrine did not appear also in presence of inhibitors of all four different types of PGE2 receptors. Western blot analysis revealed an epinephrine-induced increase in the phosphorylation of p38 MAPK and ERK that disappeared in presence of respectively PP2 and SB2020190. In addition, an inhibitory effect, similar to that of epinephrine's, was observed upon incubation with PGE2. It was concluded that epinephrine inhibits the Na+/K+-ATPase by the sequential activation of α2 adrenergic receptors, Src, p38MAPK, and ERK leading to PGE2 release.

  13. Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

    Science.gov (United States)

    Gannon, Michael K.; Holt, Jason J.; Bennett, Stephanie M.; Wetzel, Bryan R.; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.; Sawada, Geri A.; Higgins, J. William; Tombline, Gregory; Raub, Thomas J.; Detty, Michael R.

    2012-01-01

    We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells. PMID:19402665

  14. 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.

  15. Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

    Science.gov (United States)

    Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia

    2014-01-01

    Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. PMID:24379287

  16. Inhibition of the Vacuolar-like ATPase from Halobacterium saccharovorum by Thiol Reagents: Evidence for Different Functional Thiols

    Science.gov (United States)

    Hochstein, L. I.; Stanlotter, H.; Emrich, E.; Morrison, David (Technical Monitor)

    1994-01-01

    N-Ethylmaleimide (NEM) inhibited the vacuolar-like ATPase from Halobacterium saccharovorum (K(sub i) approximately 1 mM) by modifying one or more of the thiols located on the largest of the subunit. ATP protected against inhibition and coincidentally prevented NEM binding which suggested that NEM acts at or near the catalytic site. p-Chloromercuriphenylsulfonate (PCMS) also inhibited this ATPase (K(sub i) approximately 90 microM). ATP did not protect against PCMS inhibition. Dithiothreitol (DTT) partially reversed PCMS inhibition and restored approximately half of the initial activity of 90% inhibited enzyme. DTT did not restore activity of the NEM-inhibited enzyme or the PCMS-inhibited enzyme when it was subsequently incubated with NEM. The failure of ATP to protect against PCMS inhibition and the inability of DTT to restore activity of enzyme incubated in the presence of PCMS and NEM suggests these reagents react with different thiols and that the PCMS-sensitive thiol may have a structural role.

  17. A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

    Science.gov (United States)

    Stan-Lotter, Helga; Hochstein, Lawrence I.

    1989-01-01

    A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase.

  18. Changes in antioxidant status, protein concentration, acetylcholinesterase, (Na+,K+)-, and Mg2+ -ATPase activities in the brain of hyper- and hypothyroid adult rats.

    Science.gov (United States)

    Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Mourouzis, Iordanis; Varonos, Dennis; Cokkinos, Dennis; Tsakiris, Stylianos

    2005-06-01

    It is a common knowledge that metabolic reactions increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how the metabolic reactions could affect the total antioxidant status (TAS), protein concentration (PC) and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+ -ATPase in the brain of hyper- and hypothyroid adult male rats. Hyperthyroidism was induced in rats by subcutaneous administration of thyroxine (25 microg/l00 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. TAS, PC, and enzyme activities were evaluated spectrophotometrically in the homogenated brain of each animal. TAS, PC, and Mg2+ -ATPase activity were found unaffected in hyperthyroidism, while AChE and Na+,K+ -ATPase activities were reduced by 25% (p activities were found to be increased (approx. 23-30%, p activity and PC were shown to be inhibited (approx. 23-30%, p activities may reflect the different metabolic effects of hyper- and hypothyroidism. Such changes of the enzyme activities may differentially modulate the brain intracellular Mg2+, neural excitability, as well as the uptake and release of biogenic amines.

  19. Purification of the N,N'-dicyclohexylcarbodiimide-binding proteolipid of a higher plant tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Rea, P.A.; Griffith, C.J.; Sanders, D.

    1987-01-01

    The H+-ATPase of Beta vacuolar membrane (tonoplast) comprises at least three functionally distinct subunits of Mr = 67,000, 57,000, and 16,000, respectively. The hydrophobic carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the enzyme with pseudo-first order kinetics, and the concentration dependence of the reaction indicates that DCCD interacts with a single site on the enzyme to exert its inhibitory effect. The apparent pseudo-first order rate constant (k0) is reciprocally dependent on membrane protein concentration, which is expected if a large fraction of the DCCD partitions into the lipid phase. k0 has a nominal value of 1000 M-1 min-1 at a protein concentration of 250 micrograms/ml, although when phase partitioning is taken into account, the true, protein concentration-independent value of k0 is calculated to be about an order of magnitude lower. [ 14 C]DCCD primarily labels the Mr = 16,000 polypeptide of native tonoplast vesicles. Binding is venturicidin-insensitive and occurs at a rate similar to the rate of enzyme inactivation, implying that inhibition is a direct result of covalent modification of the Mr = 16,000 polypeptide. Labeling of the containing Mr = 8,000 subunit of mitochondrial F0F1-ATPase is, on the other hand, faster by a factor of 5 and totally abolished by venturicidin. These results confirm that the Mr = 16,000 polypeptide which copurifies with tonoplast H+-ATPase activity is a subunit of the enzyme. Most of the DCCD-reactive Mr = 16,000 subunit is extracted from acetone:ethanol-washed tonoplast vesicles by chloroform:methanol. [ 14 C]DCCD bound to the Mr = 16,000 polypeptide is enriched in the chloroform:methanol extract by 5-fold compared with native tonoplast and the specific activity (nmol of [ 14 C]DCCD/mg of protein) can be increased a further 37-fold by chromatography on DEAE-Sephadex

  20. OSMOTIC FRAGILITY AND Na + -K + + ATPase ACTIVITY OF ...

    African Journals Online (AJOL)

    + -K+ ATPase activity of the erythrocytes of HIV/AIDS patients. Whole blood was taken from subjects at the Human Virology Laboratory of the Nigerian Institute of Medical Research. Subjects were judged suitable for the various investigations by ...

  1. 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)

  2. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    DEFF Research Database (Denmark)

    Costa, Sara; Marek, Magdalena; Axelsen, Kristian Buhl

    2016-01-01

    and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues......) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications...

  3. Effects of stinging nettle root extracts and their steroidal components on the Na+,K(+)-ATPase of the benign prostatic hyperplasia.

    Science.gov (United States)

    Hirano, T; Homma, M; Oka, K

    1994-02-01

    The effects of organic-solvent extracts of Urtica dioica (Urticaceae) on the Na+,K(+)-ATPase of the tissue of benign prostatic hyperplasia (BPH) were investigated. The membrane Na+,K(+)-ATPase fraction was prepared from a patient with BPH by a differential centrifugation of the tissue homogenate. The enzyme activity was inhibited by 10(-4)-10(-5) M of ouabain. The hexane extract, the ether extract, the ethyl acetate extract, and the butanol extract of the roots caused 27.6-81.5% inhibition of the enzyme activity at 0.1 mg/ml. In addition, a column extraction of stinging nettle roots using benzene as an eluent afforded efficient enzyme inhibiting activity. Steroidal components in stinging nettle roots, such as stigmast-4-en-3-one, stigmasterol, and campesterol inhibited the enzyme activity by 23.0-67.0% at concentrations ranging from 10(-3)-10(-6) M. These results suggest that some hydrophobic constituents such as steroids in the stinging nettle roots inhibited the membrane Na+,K(+)-ATPase activity of the prostate, which may subsequently suppress prostate-cell metabolism and growth.

  4. Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

    Science.gov (United States)

    VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

    2016-12-01

    The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Science.gov (United States)

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase

  6. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Directory of Open Access Journals (Sweden)

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. Direct measurement of newly synthesized ATP dissociation kinetics in sarcoplasmic reticulum ATPase

    International Nuclear Information System (INIS)

    Teruel-Puche, J.; Kurzmack, M.; Inesi, G.

    1987-01-01

    Incubation of SR vesicles with Ca 2+ and ( 32 P)acetylphosphate, yields steady state levels of ( 32 P)phosphorylated enzyme (ATPase) intermediate and high concentrations of Ca 2+ in the lumen of the vesicles. At this time, addition of ADP (and EGTA to lower the Ca 2+ concentration in the medium outside the vesicles) results in single cycle formation of (γ- 32 P)ATP by transfer of ( 32 P)phosphate from the enzyme intermediate to ADP. The phosphoenzyme decay and ATP formation exhibit a fast component within the first 20 msec following addition of ADP, and a slower component reaching an asymptote in approximately 100 msec. They have now measured by a rapid filtration method the fraction of newly synthesized ATP which is bound to the enzyme, as opposed to the fraction dissociated into the medium. They find that nearly all the ATP formed during the initial burst is still bound to the enzyme within the initial 20 msec of reaction. Dissociation of newly synthesized ATP occurs then with approximately 13 sec -1 rate constant, permitting reequilibration of the system and further formation of ATP. The rate limiting effect of ATP dissociation and other partial reactions on the slow component of single cycle ATP synthesis is evaluated by appropriate kinetic simulations

  14. On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

    1990-01-16

    Washed chloroplast thylakoid membranes upon exposure to ({sup 3}H)ADP retain in tightly bound ({sup 3}H)ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg{sup 2+} results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound ({sup 3}H)ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg{sup 2+}- and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22{degree}C and of about 15 s at 37{degree}C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg{sup 2+} and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound ({sup 3}H)ADP parallels the onset of ATPase activity, although some ({sup 3}H)ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound ({sup 3}H)ADP being at a catalytic site and being replaced as this Mg{sup 2+}- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P{sub i} binding site of the enzyme-ADP-Mg{sup 2+} complex to give a form more readily activated by ATP binding at an alternative site.

  15. 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.

  16. Urea application promotes amino acid metabolism and membrane lipid peroxidation in Azolla.

    Directory of Open Access Journals (Sweden)

    Jiana Chen

    Full Text Available A pot experiment was conducted to evaluate the effect of urea on nitrogen metabolism and membrane lipid peroxidation in Azolla pinnata. Compared to controls, the application of urea to A. pinnata resulted in a 44% decrease in nitrogenase activity, no significant change in glutamine synthetase activity, 660% higher glutamic-pyruvic transaminase, 39% increase in free amino acid levels, 22% increase in malondialdehyde levels, 21% increase in Na+/K+- levels, 16% increase in Ca2+/Mg2+-ATPase levels, and 11% decrease in superoxide dismutase activity. In terms of H2O2 detoxifying enzymes, peroxidase activity did not change and catalase activity increased by 64% in urea-treated A. pinnata. These findings suggest that urea application promotes amino acid metabolism and membrane lipid peroxidation in A. pinnata.

  17. Urea application promotes amino acid metabolism and membrane lipid peroxidation in Azolla.

    Science.gov (United States)

    Chen, Jiana; Huang, Min; Cao, Fangbo; Pardha-Saradhi, P; Zou, Yingbin

    2017-01-01

    A pot experiment was conducted to evaluate the effect of urea on nitrogen metabolism and membrane lipid peroxidation in Azolla pinnata. Compared to controls, the application of urea to A. pinnata resulted in a 44% decrease in nitrogenase activity, no significant change in glutamine synthetase activity, 660% higher glutamic-pyruvic transaminase, 39% increase in free amino acid levels, 22% increase in malondialdehyde levels, 21% increase in Na+/K+- levels, 16% increase in Ca2+/Mg2+-ATPase levels, and 11% decrease in superoxide dismutase activity. In terms of H2O2 detoxifying enzymes, peroxidase activity did not change and catalase activity increased by 64% in urea-treated A. pinnata. These findings suggest that urea application promotes amino acid metabolism and membrane lipid peroxidation in A. pinnata.

  18. Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

    Science.gov (United States)

    Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

    2016-01-01

    The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF₁.

    Science.gov (United States)

    Robinson, Graham C; Bason, John V; Montgomery, Martin G; Fearnley, Ian M; Mueller, David M; Leslie, Andrew G W; Walker, John E

    2013-02-13

    The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α(DP)- and β(DP)-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β(E)-subunits. The nucleotide binding site in β(E)-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.

  20. The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

    Science.gov (United States)

    Robinson, Graham C.; Bason, John V.; Montgomery, Martin G.; Fearnley, Ian M.; Mueller, David M.; Leslie, Andrew G. W.; Walker, John E.

    2013-01-01

    The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF1 has been determined at 2.5 Å resolution. The inhibitory region of IF1 from residues 1 to 36 is entrapped between the C-terminal domains of the αDP- and βDP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F1-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic βE-subunits. The nucleotide binding site in βE-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule. PMID:23407639

  1. Fluctuation-driven directional flow of energy in biochemical cycle: Electric activation of Na,K ATPase

    Science.gov (United States)

    Yow Tsong, Tian

    1998-03-01

    Na,K ATPase is an ion pump which uses chemical bond energy of ATP to pump Na ion out of, and K ion into living cell thus maintaining ionic and osmotic balances of the cell. Both are uphill transport reactions. Surprisingly we have found that electric energy can also substitute chemical energy to fuel the pump activity. However, in this case only electric fields of certain waveforms, amplitudes, and frequencies are effective. Waveform, amplitude and frequency are three elements of signal. In other words, Na,K ATPase can recognize, process, and harvest energy from an oscillating or a fluctuating electric field to drive an endergonic reaction. The enzyme is a molecular transducer of electric signal. This report will describe electric activation experiment to define electric signal. Electric signal will then mixed with electric noise of broad power spectrum for experiment. Effect of white noise (WN) on the efficiency of Na,K ATPase will be investigated. It will be shown that WN of appropriate power level can improve the pump efficiency when a sub-optimal electric field is used. WN can also carry a sub-threshold signal to cross over the threshold. Stochastic resonance will be discussed in reference to these observations.

  2. Locally formed dopamine inhibits Na sup + -K sup + -ATPase activity in rat renal cortical tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J. (Harvard Medical School, Boston, MA (USA) Karolinska Institute, Stockholm (Sweden))

    1988-10-01

    Dopamine, generated locally from L-dopa, inhibits Na{sup +}-K{sup +}-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na{sup +}-K{sup +}-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate ({dot Q}o{sub 2}) and {sup 86}Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive {dot Q}o{sub 2} or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive {dot Q}o{sub 2} in a concentration-dependent manner, with half-maximal inhibition (K{sub 0.5}) of 5 {times} 10{sup {minus}7} M and a maximal inhibition of 14.1 {plus minus} 1.5% at 10{sup {minus}4}M. L-Dopa also blunted the nystatin-stimulated {dot Q}o{sub 2} in a concentration-dependent manner, indicating the L-dopa directly inhibits Na{sup +}-K{sup +}-ATPase activity and not sodium entry. Ouabain-sensitive {sup 86}Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} and {sup 86}Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na{sup +}-K{sup +}-ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner.

  3. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  4. 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.

  5. 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

  6. Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

    Science.gov (United States)

    Nedukha, Olena

    2016-07-01

    The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

  7. 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...

  8. 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.

  9. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    Science.gov (United States)

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  10. Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

    Science.gov (United States)

    Zivkovic, Danica; Créton, Robbert; Zwaan, Gideon; de Bruijn, Willem C; Dohmen, M René

    1990-11-01

    During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca 2+ /Mg 2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca 2+ /Mg 2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca 2+ -stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.

  11. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost

    2016-01-01

    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... to the cerebellar Na(+) and K(+) gradients....... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...

  12. Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation.

    Science.gov (United States)

    Huang, Chun-Yen; Chao, Pei-Lin; Lin, Hui-Chen

    2010-03-01

    The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model. Copyright 2009 Elsevier B.V. All rights reserved.

  13. Structural aspects of proton-pumping ATPases

    NARCIS (Netherlands)

    Walker, J. E.; Fearnley, I. M.; Lutter, R.; Todd, R. J.; Runswick, M. J.

    1990-01-01

    ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of alpha 3 beta 3 gamma 1 delta 1 epsilon 1

  14. K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion.

    Science.gov (United States)

    Garçon, D P; Masui, D C; Mantelatto, F L M; McNamara, J C; Furriel, R P M; Leone, F A

    2007-05-01

    To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.

  15. Microtubule-dependent relocation of branchial V-H+-ATPase to the basolateral membrane in the Pacific spiny dogfish (Squalus acanthias): a role in base secretion.

    Science.gov (United States)

    Tresguerres, Martin; Parks, Scott K; Katoh, Fumi; Goss, Greg G

    2006-02-01

    We have previously shown that continuous intravenous infusion of NaHCO3 for 24 h ( approximately 1000 micromol kg(-1) h(-1)) results in the relocation of V-H+-ATPase from the cytoplasm to the basolateral membrane in the gills of the Pacific dogfish. To further investigate this putative base-secretive process we performed similar experiments with the addition of colchicine, an inhibitor of cytoskeleton-dependent cellular trafficking processes. Blood pH and plasma total CO2 were significantly higher in the colchicines-treated, HCO3- -infused fish compared with fish infused with HCO3- alone. The effect of colchicine was highest after 24 h of infusion (8.33+/-0.06 vs 8.02+/-0.03 pH units, 15.72+/-3.29 vs 6.74+/-1.34 mmol CO2 l(-1), N=5). Immunohistochemistry and western blotting confirmed that colchicine blocked the transit of V-H+-ATPase to the basolateral membrane. Furthermore, western blotting analyses from whole gill and cell membrane samples suggest that the short-term (6 h) response to alkaline stress consists of relocation of V-H+-ATPases already present in the cell to the basolateral membrane, while in the longer term (24 h) there is both relocation of preexistent enzyme and upregulation in the synthesis of new units. Our results strongly suggest that cellular relocation of V-H+-ATPase is necessary for enhanced HCO3- secretion across the gills of the Pacific dogfish.

  16. Effect of interaction of heavy metals on (Na+-K+) ATPase and uptake of 3H-DA and 3H-NA in rat brain synaptosomes

    International Nuclear Information System (INIS)

    Chandra, S.V.; Murthy, R.C.; Husain, T.; Bansal, S.K.

    1984-01-01

    The effect of interaction of Mn 2+ , Pb 2+ and CD 2+ on (Na + -K + ) ATPase and uptake of labelled dopamine ( 3 H-DA) and labelled noradrenaline ( 3 H-NA) were studied in vitro in rat brain synaptosomes. The inhibition of (Na + -K + )ATPase by Pb 2+ + Cd 2+ alone was concentration dependent, however, Mn 2+ had almost no effect on the activity of this enzyme. Interaction of Cd 2+ with either Pb 2+ or Mn 2+ was almost powerful in inhibiting the activity of synaptosomal transport ATPase. Lower concentrations of Pb 2+ increased while higher concentrations inhibited synaptosomal uptake of 3 H-DA and 3 H-NA. Lower concentrations of CD 2+ increased the uptake of 3 H-DA while at concentrations of 100 μM, the uptake was inhibited, this metal had strong inhibitory effect on the uptake of 3 H-NA. Mn 2+ had inhibited the uptake of labelled amines. Interaction of Mn 2+ with Pb 2+ or Cd 2+ produced inhibition on the uptake of 3 H-DA and 3 H-NA. The results of the uptake of biogenic amines in the presence of metal ions apparently had no correlation with the activity og (Na + -K + ) ATPase which is involved in the active transport of cations across cell membranes. (author)

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

    Directory of Open Access Journals (Sweden)

    Lu-Ning Sun

    2017-09-01

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

  18. 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...

  19. Protein import into chloroplasts requires a chloroplast ATPase

    International Nuclear Information System (INIS)

    Pain, D.; Blobel, G.

    1987-01-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [ 35 S]methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H + , K + , Na + , or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors

  20. 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.

  1. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... resolution 3D structure the mechanism behind is only poorly understood. This thesis aimed at illuminating the autoinhibitory mechanism in plant and yeast PM H+-ATPases and below some of our main findings will be highlighted. The two terminal domains of the PM H+-ATPases have several amino acid residues...... that can be phosphorylated, and it has been demonstrated that these phosphorylation sites in both plant and yeast are highly involved in the regulation of terminal autoinhibition. In this study we used a phylogenetic analysis to investigate the evolutionary development of these phosphorylation sites...

  2. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

    Science.gov (United States)

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B; Holding, Andrew N; Montgomery, Martin G; Fearnley, Ian M; Walker, John E

    2015-05-22

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking*

    Science.gov (United States)

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B.; Holding, Andrew N.; Montgomery, Martin G.; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. PMID:25851905

  4. Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs of Lymnaea stagnalis and Bithynia tentaculata (Mollusca)

    OpenAIRE

    Zivkovic, Dana; Créton, Robbert; Zwaan, Gideon; Bruijn, Wim; Dohmen, M.René

    1990-01-01

    textabstractDuring extrusion of the first polar body in eggs of Lymnaea stagnalis and Bithynia tentaculata a localized Ca2+ /Mg2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs of Lymnaea it was found only in the vegetal hemisphere, whereas in Bithynia eggs it was localized...

  5. Inhibition of the ATPase from Halobacterium Saccharovorum by Thiol Inhibitors: Evidence for the Presence of More Than One Essential Cysteinyl Residue

    Science.gov (United States)

    Hochstein, Lawrence I.; Emrich, Errol; Stan-Lotter, Helga; DeVincenzi, Donald L. (Technical Monitor)

    1995-01-01

    The vacuolar-like ATPase from Halobacterium saccha vorum is inhibited by N-ethylmaleimide and p-chloromercudphenylsulfonate. The failure of adenine nucleotides to protect against p-chloromercuriphenyisulfonate inhibition, of p-chloromercuriphenylsulfonate to protect against N-ethylmaleimide inhibition, and the difference in the temperature dependence of inactivation infers that the enzyme contains at least two thiols that are essential for enzyme activity. CNBr cleavage of C-14-N-ethylmaleimide labeled subunit results in two radioactive peptides that locates the N-ethylmaleimide-reactive cysteinyl residue as cysteine-262 in the H. salinarium sequence.

  6. 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

  7. 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...

  8. 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...

  9. 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...

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  11. Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection

    Directory of Open Access Journals (Sweden)

    Alison W. Ashbrook

    2016-05-01

    Full Text Available Chikungunya virus (CHIKV is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. There are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. We conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium ATPase, as a potent inhibitor of CHIKV infection. Treatment of human cells with digoxin or a related cardiac glycoside, ouabain, resulted in a dose-dependent decrease in infection by CHIKV. Inhibition by digoxin was cell type-specific, as digoxin treatment of either murine or mosquito cells did not diminish CHIKV infection. Digoxin displayed antiviral activity against other alphaviruses, including Ross River virus and Sindbis virus, as well as mammalian reovirus and vesicular stomatitis virus. The digoxin-mediated block to CHIKV and reovirus infection occurred at one or more postentry steps, as digoxin inhibition was not bypassed by fusion of CHIKV at the plasma membrane or infection with cell surface-penetrating reovirus entry intermediates. Selection of digoxin-resistant CHIKV variants identified multiple mutations in the nonstructural proteins required for replication complex formation and synthesis of viral RNA. These data suggest a role for the sodium-potassium ATPase in promoting postentry steps of CHIKV replication and provide rationale for modulation of this pathway as a broad-spectrum antiviral strategy.

  12. [3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

    International Nuclear Information System (INIS)

    Shoemaker, D.G.; Lauf, P.K.

    1983-01-01

    The interaction of the cardiac glycoside [ 3 H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [ 3 H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [ 3 H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [ 3 H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor

  13. 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...

  14. 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....

  15. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2008-01-01

    of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K+. Low-affinity ATP interaction with the enzyme was strongly reduced following CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite...

  16. Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin.

    Science.gov (United States)

    Li, Sisi; Yen, Linda; Pastor, William A; Johnston, Jonathan B; Du, Jiamu; Shew, Colin J; Liu, Wanlu; Ho, Jamie; Stender, Bryan; Clark, Amander T; Burlingame, Alma L; Daxinger, Lucia; Patel, Dinshaw J; Jacobsen, Steven E

    2016-08-30

    Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.

  17. The Wheat E Subunit of V-Type H+-ATPase Is Involved in the Plant Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    Xiao-Hong Zhang

    2014-09-01

    Full Text Available The vacuolar type H+-ATPase (V-type H+-ATPase plays important roles in establishing an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.

  18. 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.

  19. 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.

  20. (/sup 3/H)ouabain localization of Na-K ATPase in the epithelium of rabbit ciliary body pars plicata

    Energy Technology Data Exchange (ETDEWEB)

    Usukura, J.; Fain, G.L.; Bok, D.

    1988-04-01

    The secretion of the aqueous humor has been proposed to occur as the result of active Na+ transport by a ouabain-sensitive Na-K ATPase. We have examined the localization of this enzyme in the epithelium of rabbit ciliary body pars plicata using (3H)ouabain autoradiography. Single ciliary processes were isolated and incubated in Ringer containing (3H)ouabain. Processes were then rapidly frozen, freezedried, sectioned and exposed for autoradiography. In the light microscope, silver grains were found predominantly over the nonpigmented epithelial cells. In the electron microscope, grains could be localized for the most part to the interdigitations of the nonpigmented cell basolateral membrane. Label could also be observed at a much lower density above other membranes and above the pigmented and nonpigmented cell cytoplasm. No label was found in sections of control tissue which had been incubated in (3H)ouabain with an excess of cold ouabain. To show that the (3H)ouabain had free access to all of the membrane surfaces within the epithelium, in parallel experiments we incubated isolated processes in horseradish peroxidase. Our experiments suggest that most of the active Na+ transport in ciliary body epithelium occurs across the basolateral membrane of nonpigmented cells into the posterior chamber. Furthermore, the placement of the Na-K ATPase within the narrow membrane infoldings of the interdigitations is consistent with a role for this enzyme in water transport and the production of the aqueous.

  1. The effect of X-irradiation on Na-K ATPase and cation distribution in rabbit lens

    International Nuclear Information System (INIS)

    Matsuda, H.; Giblin, F.J.; Reddy, V.N.

    1982-01-01

    The Na-K ATPase activity of rabbit lens was measured at various times after exposure to a single dose of 2000 rads of X-ray and was compared with that in contralateral control eyes. A decrease in enzyme activity in both whole lens and in isolated capsule-epithelium was first observed 3 to 4 weeks after irradiation and became increasingly marked at 7.5 weeks after X-ray. These findings are consistent with our earlier observations that active transport of cations is reduced in these lenses and support the view that loss of membrane ATPase is responsible for the impairment of the cation pump in X-irradiated lenses. Despite a significant loss of the enzyme, X-irradiated lenses were able to maintain near normal levels of total cations (Na+ + K+), thus accounting for their normal hydration. The results of the changes in lens Na+ and K+ levels revealed that between 4 and 7.5 weeks after X-ray, the gain in Na+ was compensated by an equivalent loss of K+. A breakdown of this relationship of 1:1 exchange of Na+ for K+ is accompanied by a disproportionate increase in Na+ and water

  2. Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Ng, Y.C.; Akera, T.

    1987-05-01

    In partially purified Na+-K+-adenosinetriphosphatase (ATPase) obtained from ferret heart, ouabain produced a monophasic inhibition curve; however, the curve spanned over 5 logarithmic units, indicating the presence of more than one classes of enzyme. (/sup 3/H)ouabain binding studies revealed high-and low-affinity binding sites in approximately equal abundance, with apparent dissociation constants of 10 and 230 nM, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphoenzyme formed from (gamma-/sup 32/P)ATP showed two distinct K+-sensitive bands of approximately 100,000 molecular weight. Phosphoenzyme formation from the high-molecular-weight alpha(+) form was selectively inhibited by N-ethylmaleimide. Ouabain caused a 50% inhibition of phosphorylation of the alpha(+) form at 40 nM and the lower-molecular-weight alpha form at 300 nM. In papillary muscle preparations, 1-30 nM ouabain produced a modest positive inotropic effect that reached an apparent plateau at 30 nM. Further increases in ouabain concentrations, however, produced additional and prominent inotropic effects at 0.1-10 microM. These results indicate for the first time in cardiac muscle that the high- and low-affinity ouabain binding sites are associated with the alpha(+) and alpha forms of the Na+-K+-ATPase, respectively, and that binding of ouabain to either of these sites causes enzyme inhibition and the positive inotropic effect.

  3. Cadmium induced inhibition of Na sup + /K sup + ATPase activity in tissues of crab Scylla serrata (Forskal)

    Energy Technology Data Exchange (ETDEWEB)

    Dhavale, D.M. (RJ College, Bombay (India)); Masurekar, V.B. (Institute of Science, Bombay (India)); Giridhar, B.A. (Univ. of Utah, Salt Lake City (USA))

    1988-06-01

    Heavy metals discharged from industries these days are a major source of pollution which has become threat to all forms of life. A measure of metabolism may be a most sensitive parameter since it integrates many factors such as enzyme activity, biochemical contents and physiological response. The ability to accurately characterize enzymes with respect to their distribution and kinetics makes them attractive indices of stress. The concept of Na{sup +}/K{sup +}-ATPase being intimately involved in active transport of ions across biological membranes has gained wide acceptance in recent years. Interference with osmoregulation may restrict the animal's ability to adapt to salinity changes. Several studies have shown that crustacenas are highly sensitive to metal pollutants, particularly when acclimated to low salinities. The present study was initiated to compare the characteristics of Na{sup +}/K{sup +}-ATPase activity in hepatopancreas and gills of crabs Scylla serrata (Forskal) exposed to acute and sublethal concentrations of cadmium chloride for defined periods with those from undosed animals.

  4. 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...

  5. 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...

  6. 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....

  7. 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.

  8. 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.

  9. Kinetic discrimination of self/non-self RNA by the ATPase activity of RIG-I and MDA5.

    Science.gov (United States)

    Louber, Jade; Brunel, Joanna; Uchikawa, Emiko; Cusack, Stephen; Gerlier, Denis

    2015-07-28

    The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5'-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5-2 kb long dsRNA as canonical agonists, respectively. However, in vitro, they can bind to many RNA species, while in cells there is an activation threshold. As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial. A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged. The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response

  10. 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...

  11. 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

  12. 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 ...

  13. Changes in erythrocyte ATPase activity under different pathological ...

    African Journals Online (AJOL)

    Changes in erythrocyte ATPase activity under different pathological conditions. Ali A Kherd, Nawal Helmi, Khadijah Saeed Balamash, Taha A Kumosani, Shareefa A AL-Ghamdi, Qari M, Etimad A Huwait, Soonham S Yaghmoor, Alaama Nabil, Maryam A AL-Ghamdi, Said S Moselhy ...

  14. The Kdp-ATPase system and its regulation

    Indian Academy of Sciences (India)

    2007-03-15

    Mar 15, 2007 ... K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E. coli, the KdpATPase (comprising of the KdpFABC complex), ...

  15. Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

    NARCIS (Netherlands)

    Jackson, R.L.; Verkleij, A.J.; Zoelen, E.J.J. van; Lane, L.K.; Schwartz, A.; Deenen, L.L.M. van

    Purified lamb kidney Na+, K+-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the Mτ- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles

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

    African Journals Online (AJOL)

    2016-11-09

    Nov 9, 2016 ... Spodoptera exigua larval development by silencing chitin synthase gene with RNA interference. Bull. Entomol. Res. 98:613-619. Dow JAT (1999). The Multifunctional Drosophila melanogaster V-. ATPase is encoded by a multigene family. J. Bioenerg. Biomembr. 31:75-83. Fire A, Xu SQ, Montgomery MK, ...

  17. Selective induction of high-ouabain-affinity isoform of Na sup + -K sup + -ATPase by thyroid hormone

    Energy Technology Data Exchange (ETDEWEB)

    Haber, R.S.; Loeb, J.N. (Columbia Univ., New York, NY (USA))

    1988-12-01

    The administration of thyroid hormone is known to result in an induction of the Na{sup +}-K{sup +}-adenosinetriphosphatase (Na{sup +}-K{sup +}-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na{sup +}-K{sup +}-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3{prime},5-triiodo-L-thyronine (T{sub 3}) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na{sup +}-K{sup +}-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K{sub I}s) for ouabain of {approximately}10{sup {minus}7} and 10{sup {minus}4} M, respectively. Measurement of the specific binding of ({sup 3}H)ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K{sub d}) of slightly less than 10{sup {minus}7}M, whose maximal binding capacity was increased by T{sub 3} treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T{sub 3} treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na{sup +}-K{sup +}-ATPase by ouabain-dependent phosphorylation from ({sup 32}P)orthophosphate confirmed that T{sub 3} treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na{sup +}-K{sup +}-ATPase are subject to selective hormonal induction.

  18. Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

    International Nuclear Information System (INIS)

    Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

    1987-01-01

    In this work, we examined occlusion of 22 Na+ and 86 Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of 22 Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of 22 Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by 32 P incorporation from [gamma- 32 P]CrATP. Maximum capacity for phosphorylation from [gamma- 32 P]CrATP was 6 nmol/mg of protein and equal to capacities for binding of [48V]vanadate and [ 3 H]ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport

  19. Occlusion of /sup 22/Na+ and /sup 86/Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

    1987-08-05

    In this work, we examined occlusion of /sup 22/Na+ and /sup 86/Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of /sup 22/Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of /sup 22/Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by /sup 32/P incorporation from (gamma-/sup 32/P)CrATP. Maximum capacity for phosphorylation from (gamma-/sup 32/P)CrATP was 6 nmol/mg of protein and equal to capacities for binding of (48V)vanadate and (/sup 3/H)ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport.

  20. 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.

  1. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

  2. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  3. 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.

  4. Effects of lead on Na+, K+-ATPase and hemolymph ion concentrations in the freshwater mussel Elliptio complanata

    Science.gov (United States)

    Mosher, Shad; Cope, W. Gregory; Weber, Frank X.; Shea, Damian; Kwak, Thomas J.

    2012-01-01

    Freshwater mussels are an imperiled fauna exposed to a variety of environmental toxicants such as lead (Pb) and studies are urgently needed to assess their health and condition to guide conservation efforts. A 28-day laboratory toxicity test with Pb and adult Eastern elliptio mussels (Elliptio complanata) was conducted to determine uptake kinetics and to assess the toxicological effects of Pb exposure. Test mussels were collected from a relatively uncontaminated reference site and exposed to a water-only control and five concentrations of Pb (as lead nitrate) ranging from 1 to 245 mu g/L in a static renewal test with a water hardness of 42 mg/L. Endpoints included tissue Pb concentrations, hemolymph Pb and ion (Na+, K+, Cl-, Ca2+) concentrations, and Na+, K+-ATPase enzyme activity in gill tissue. Mussels accumulated Pb rapidly, with tissue concentrations increasing at an exposure-dependent rate for the first 2 weeks, but with no significant increase from 2 to 4 weeks. Mussel tissue Pb concentrations ranged from 0.34 to 898 mu g/g dry weight, were strongly related to Pb in test water at every time interval (7, 14, 21, and 28 days), and did not significantly increase after day 14. Hemolymph Pb concentration was variable, dependent on exposure concentration, and showed no appreciable change with time beyond day 7, except for mussels in the greatest exposure concentration (245 mu g/L), which showed a significant reduction in Pb by 28 days, suggesting a threshold for Pb binding or elimination in hemolymph at concentrations near 1000 mu g/g. The Na+, K+-ATPase activity in the gill tissue of mussels was significantly reduced by Pb on day 28 and was highly correlated with tissue Pb concentration (R2 = 0.92; P = 0.013). The Na+, K+-ATPase activity was correlated with reduced hemolymph Na+ concentration at the greatest Pb exposure when enzyme activity was at 30% of controls. Hemolymph Ca2+ concentration increased significantly in mussels from the greatest Pb exposure and may

  5. The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly

    Energy Technology Data Exchange (ETDEWEB)

    Her, Joonyoung [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of); Chung, In Kwon, E-mail: topoviro@yonsei.ac.kr [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Identification of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. Black-Right-Pointing-Pointer NVL2 associates with catalytically active telomerase via an interaction with hTERT. Black-Right-Pointing-Pointer NVL2 is a telomerase component essential for holoenzyme assembly. Black-Right-Pointing-Pointer ATP-binding activity of NVL2 is required for hTERT binding and telomerase assembly. -- Abstract: Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.

  6. Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2.

    Science.gov (United States)

    Vestergaard, Anna L; Coleman, Jonathan A; Lemmin, Thomas; Mikkelsen, Stine A; Molday, Laurie L; Vilsen, Bente; Molday, Robert S; Dal Peraro, Matteo; Andersen, Jens Peter

    2014-04-08

    P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  8. Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

    Science.gov (United States)

    Han, Xiaolin; Liu, Ping; Gao, Baoquan; Wang, Haofeng; Duan, Yafei; Xu, Wenfei; Chen, Ping

    2015-07-01

    Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes' α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5' untranslated region (UTR) of 317 bp, and 3' UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-05-30

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

  10. Enzyme-Catalyzed Transetherification of Alkoxysilanes

    Directory of Open Access Journals (Sweden)

    Peter G. Taylor

    2013-01-01

    Full Text Available We report the first evidence of an enzyme-catalyzed transetherification of model alkoxysilanes. During an extensive enzymatic screening in the search for new biocatalysts for silicon-oxygen bond formation, we found that certain enzymes promoted the transetherification of alkoxysilanes when tert-butanol or 1-octanol were used as the reaction solvents.

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

    Directory of Open Access Journals (Sweden)

    Lijun Catherine Liu

    2016-09-01

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

  12. Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase.

    Science.gov (United States)

    Petrov, V V

    2015-01-01

    The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp®Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34-95% of the wild-type level) showed activity high enough (35-108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5-M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.

  13. Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with 125I-derivatives of cymarin and digitoxigenin

    International Nuclear Information System (INIS)

    Lowndes, J.M.

    1988-01-01

    NaK-ATPase is an enzyme which maintains Na + and K + gradients across the plasma membrane of eukaryotic cells, and is specifically inhibited by cardiac glycosides. The cardiac glycoside binding site is located primarily on the catalytic α subunit but the glycoprotein β and proteolipid-γ subunits may also contribute to the structure of the site. In order to label the cardiac glycoside binding site of human erythrocytes, four photoaffinity ligands with very high specific radioactivity were synthesized. The compounds, which are abbreviated [ 125 I]AISC, [ 125 I]AIPP-GluD, [ 125 I]AIPP-GalD and [ 125 I]IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the α, β, and proteolipid-γ subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, [ 125 I]I-TASC, was synthesized; this compound had the same structure as [ 125 I]AISC except that a light-sensitive azide group was replaced with a hydroxyl group. Competitive binding assays with cymarin against 0.2 nM [ 125 I]I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct [ 125 I]I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K D of 54 pM and a B max of 23 fmol/mg protein

  14. Influence of cadmium on ketamine-induced anesthesia and brain microsomal Na[sup +], K[sup +]-ATPase in mice

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Y.; Sangiah, S. (Oklahoma State Univ., Stillwater, OK (United States))

    1994-10-01

    Cadmium is a rare metallic element, present in almost all types of food. Shellfish, wheat and rice accumulate very high amounts. Occupational and environmental pollutants are the main sources of cadmium exposure. Cadmium has a very long biologic half-life. Exposure to Cadmium causes anemia, hypertension, hepatic, renal, pulmonary and cardiovascular disorders as well as being a possible mutagen, teratogen and carcinogen. Acute cadmium treatment increased the hexobarbital sleeping time and inhibited hepatic microsomal drug metabolism due to a decrease in cytochrome P[sub 450] content. Cadmium potentiated ethanol-induced sleep in a dose-dependent manner. Cadmium has been shown to inhibit brain microsomal Na[sup +], K[sup +]-ATPase activity in vitro and in vivo. Cadmium and ethanol additively inhibited brain Na[sup +], K[sup +]-ATPase. This might be a direct interaction between cadmium and ethanol in the central nervous system. Ketamine is an intravenous anesthetic agent. It acts on central nervous system and produces [open quotes]dissociative anaesthesia.[close quotes] Ketamine provides adequate surgical anesthesia and is used alone in humans and/or combination with xylazine, an [alpha][sub 2]-adrenergic agonist in animals. It produces CNS depression, analgesia, amnesia, immobility and a feeling of dissociation from the environment. Ketamine is a non-competitive antagonist of the NMDA subset of the glutamate receptor. This perhaps results in an increase in neuronal activity leading to disorganization of normal neurotransmission and produces dissociative anesthetic state. Because it is different from most other anesthetics, ketamine may be expected to have a unique effect on brain biochemical parameters and enzymes. The purpose of this study was to examine the interactions between cadmium and ketamine on the central nervous system and ATPase, in an attempt to further understand the mechanism of action. 12 refs., 3 figs.

  15. Fourier transform infrared spectroscopic studies of the secondary structure and thermal denaturation of CaATPase from rabbit skeletal muscle

    Science.gov (United States)

    Jaworsky, Mark; Brauner, Joseph W.; Mendelsohn, Richard

    Fourier transform i.r. spectroscopy has been used to monitor structural alterations induced by thermal denaturation of the intrinsic membrane protein CaATPase in aqueous media. The protein has been isolated, purified and studied in five forms: (i) In its native lipid environment after isolation from rabbit sarcoplasmic reticulum, both in H 2O and D 2O suspensions. (ii) After both mild and extensive tryptic digestion has cleaved those residues external to the membrane bilayer. (iii) Reconstituted in vesicle form with bovine brain sphingomyelin. Fourier deconvolution techniques have been used to enhance the resolution of the intrinsically overlapped Amide I and Amide II spectral regions. Large spectral alterations apparent in the deconvoluted spectra occur in these regions upon thermal denaturation of the protein which are consistent with the formation of a large proportion of β-antiparallel sheet form. The alteration parallels the loss in ATPase activity. A mild tryptic digestion increases slightly the proportion of α-helix and/or random coil secondary structure. A thermal transition to a form containing a high proportion of β structure is still evident. Extensive tryptic digestion nearly abolishes the alpha helical plus random coil secondary structure, while producing a high proportion of β form which is resistant to further thermally induced structural alterations. Studies of CaATPase reconstituted into vesicles with bovine brain sphingomyelin reveal a higher proportion of β structure than the native enzyme, with further introduction of β structure on thermal denaturation. Both the utility of deconvolution techniques and the necessity for caution in their application are apparent from the current experiments.

  16. Mechanism of photoinactivation of plant plasma membrane ATPases

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  17. Structural and functional characterization of P4-ATPase lipid flippases

    DEFF Research Database (Denmark)

    Ulstrup, Jakob

    2018-01-01

    to its much larger substrate and how the mechanism allowing the transport unfolds. This is one of the central questions in the field known as the “giant substrate problem”. To this date, no structural information of P4-ATPases is available. The focus of this thesis is divided into two projects, both...... focusing on P4-ATPases from the yeast organism Saccharomyces cerevisiae: I. The structural characterization of the flippase Drs2p in complex with its auxiliary subunit Cdc50p. II. Establishing a protocol for obtaining a homogenous sample of the flippase Neo1p suitable for biochemical characterization...... and substrate identification. Part I was performed using X-ray crystallography and single-particle electron microscopy as the main methods. A 3D envelope was obtained by cryo-EM extending to a resolution of 4.4 Å. This envelope reveals the first structural insight of the conformational organization of the Drs2p...

  18. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  19. Protein import into chloroplasts requires a chloroplast ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pain, D.; Blobel, G.

    1987-05-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-01

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

  1. Radiation inactivation analysis of chloroplast CF0-CF1 ATPase

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  2. 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.

  3. 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....

  4. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  5. 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.

  6. The influence of early life interventions on olfactory memory related to palatable food, and on oxidative stress parameters and Na+/K+-ATPase activity in the hippocampus and olfactory bulb of female adult rats.

    Science.gov (United States)

    Noschang, Cristie; Krolow, Rachel; Arcego, Danusa M; Laureano, Daniela; Fitarelli, Luiza D; Huffell, Ana Paula; Ferreira, Andréa G K; da Cunha, Aline A; Machado, Fernanda Rossato; Wyse, Angela T S; Dalmaz, Carla

    2012-08-01

    The effects of neonatal handling and the absence of ovarian hormones on the olfactory memory related to a palatable food in adulthood were investigated. Oxidative stress parameters and Na+/K+-ATPase activity in the hippocampus and olfactory bulb of adult pre-puberty ovariectomized female rats handled or not in the neonatal period were also evaluated. Litters were non-handled or handled (10 min/day, days 1-10 after birth). Females from each litter were divided into: OVX (subjected to ovariectomy), sham, and intact. When adults, olfactory memory related to a palatable food (chocolate) was evaluate using the hole-board olfactory task. Additionally, oxidative stress parameters and Na+/K+-ATPase activity were measured in the hippocampus and olfactory bulb. No difference between groups was observed considering olfactory memory evaluation. Neonatal handled rats presented an increase in Na+/K+-ATPase activity in the hippocampus and in the olfactory bulb, compared to non-handled ones. Considering the surgical procedure, there was a decrease in Na+/K+-ATPase and catalase activities in sham and OVX groups, compared to intact animals in the olfactory bulb. We concluded that olfactory memory related to a palatable food in adulthood was not affected by neonatal handling or by pre-puberty surgery, with or without removal of ovaries. The difference observed between groups in catalase and Na+/K+-ATPase activity does not seem to be related to the olfactory memory. Additionally, the increase in Na+/K+-ATPase activity (an enzyme that maintains the neurochemical gradient necessary for neuronal excitability) induced by neonatal handling may be related to neuroplastic changes in the hippocampus and olfactory bulb.

  7. Reduced levels of skeletal muscle Na+K+ -ATPase in McArdle disease

    Science.gov (United States)

    Haller, R. G.; Clausen, T.; Vissing, J.; Blomqvist, C. G. (Principal Investigator)

    1998-01-01

    We evaluated the hypothesis that impaired sarcolemmal function associated with exaggerated potassium release, impaired potassium uptake, or both may contribute to exertional fatigue and abnormal circulatory responses to exercise in McArdle disease (MD). The cellular mechanism of exertional fatigue and muscle injury in MD is unknown but likely involves impaired function of the ATPases that couple ATP hydrolysis to cellular work, including the muscle sodium potassium pump (Na+K+-ATPase). However, the concentration of muscle Na+K+ pumps in MD is not known, and no studies have related exercise increases in blood potassium concentrations to muscle Na+K+ pump levels. We measured muscle Na+K+ pumps (3H-ouabain binding) and plasma K+ in response to 20 minutes of cycle exercise in six patients with MD and in six sex-, age-, and weight-matched sedentary individuals. MD patients had lower levels of 3H-ouabain binding (231 +/- 18 pmol/g w.w., mean +/- SD, range, 210 to 251) than control subjects (317 +/- 37, range, 266 to 371, p Na+K+ pump levels are low in MD patients compared with healthy subjects and identify a limitation of potassium reuptake that could result in sarcolemmal failure during peak rates of membrane activation and may promote exaggerated potassium-activated circulatory responses to submaximal exercise. The mechanism of the low Na+K+ pump concentrations in MD is unknown but may relate to deconditioning or to disruption of a close functional relationship between membrane ion transport and glycolysis.

  8. Direct evidence of impaired neuronal Na/K-ATPase pump function in alternating hemiplegia of childhood.

    Science.gov (United States)

    Simmons, Christine Q; Thompson, Christopher H; Cawthon, Bryan E; Westlake, Grant; Swoboda, Kathryn J; Kiskinis, Evangelos; Ess, Kevin C; George, Alfred L

    2018-03-19

    Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential

  9. Reduced levels of skeletal muscle Na+K+ -ATPase in McArdle disease

    Science.gov (United States)

    Haller, R. G.; Clausen, T.; Vissing, J.; Blomqvist, C. G. (Principal Investigator)

    1998-01-01

    We evaluated the hypothesis that impaired sarcolemmal function associated with exaggerated potassium release, impaired potassium uptake, or both may contribute to exertional fatigue and abnormal circulatory responses to exercise in McArdle disease (MD). The cellular mechanism of exertional fatigue and muscle injury in MD is unknown but likely involves impaired function of the ATPases that couple ATP hydrolysis to cellular work, including the muscle sodium potassium pump (Na+K+-ATPase). However, the concentration of muscle Na+K+ pumps in MD is not known, and no studies have related exercise increases in blood potassium concentrations to muscle Na+K+ pump levels. We measured muscle Na+K+ pumps (3H-ouabain binding) and plasma K+ in response to 20 minutes of cycle exercise in six patients with MD and in six sex-, age-, and weight-matched sedentary individuals. MD patients had lower levels of 3H-ouabain binding (231 +/- 18 pmol/g w.w., mean +/- SD, range, 210 to 251) than control subjects (317 +/- 37, range, 266 to 371, p < 0.0004), higher peak increases in plasma potassium in response to 45 +/- 7 W cycle exercise (MD, 1.00 +/- 0.15 mmol/L; control subjects, 0.48 +/- 0.09; p < 0.0001), and mean exercise heart rate responses to exercise that were 45 +/- 12 bpm greater than control subjects. Our results indicate that Na+K+ pump levels are low in MD patients compared with healthy subjects and identify a limitation of potassium reuptake that could result in sarcolemmal failure during peak rates of membrane activation and may promote exaggerated potassium-activated circulatory responses to submaximal exercise. The mechanism of the low Na+K+ pump concentrations in MD is unknown but may relate to deconditioning or to disruption of a close functional relationship between membrane ion transport and glycolysis.

  10. Properties of Na+/K+ ATPase and alkaline phosphatase alter during spontaneous and radiation-induced leukemogenesis in mice

    International Nuclear Information System (INIS)

    Gonta-Grabiec, K.; Rossowski, W.

    1986-01-01

    Properties are characterized of Na + /K + ATPase and alkaline phosphatase in thymocytes or thymoblasts from mice of two strains: AKR in which thymoma developed spontaneously, and C57Bl in which the development was induced by X-irradiation (total dose: 5.4 Gy in 3 fractions). It was found that before thymoma could be discerned morphologically the properties of the two enzymes changed. There was a decrease in 86 Rb uptake and in the rate of ATP hydrolysis per cell (both strains) as well as an increase in alkaline phosphatase activity per cell (C57Bl mice). In both spontaneous and radiation-induced thymomas 86 Rb uptake, ATP hydrolysis and 3 H-ouabain binding per cell were higher than in normal thymuses. Likewise, alkaline phosphatase activity per cell was higher in the thymomas than in the thymuses; this increase was accompanied by the appearance of additional isoenzyme(s) (1 in AKR, 2 in C57Bl). These changes were compared with cAMP content and 3 H-thymidine incorporation, taken as indicators of the proliferative activity, and their high correlation in both AKR and C57Bl mice allowed to distinguish a pre-leukemic period. In that period thymoblasts clearly differed from the normal ones in Na + /K + ATPase and alkaline phosphatase properties as well as proliferation, although the morphology of the thymus was still unchanged. (author)

  11. [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.

  12. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

    2009-01-01

    ,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

  13. 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.

  14. Enzymes in CO2 Capture

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gladis, Arne; Thomsen, Kaj

    The enzyme Carbonic Anhydrase (CA) can accelerate the absorption rate of CO2 into aqueous solutions by several-fold. It exist in almost all living organisms and catalyses different important processes like CO2 transport, respiration and the acid-base balances. A new technology in the field...... of carbon capture is the application of enzymes for acceleration of typically slow ternary amines or inorganic carbonates. There is a hidden potential to revive currently infeasible amines which have an interesting low energy consumption for regeneration but too slow kinetics for viable CO2 capture. The aim...... of this work is to discuss the measurements of kinetic properties for CA promoted CO2 capture solvent systems. The development of a rate-based model for enzymes will be discussed showing the principles of implementation and the results on using a well-known ternary amine for CO2 capture. Conclusions...

  15. Assessment of D-methionine protecting cisplatin-induced otolith toxicity by vestibular-evoked myogenic potential tests, ATPase activities and oxidative state in guinea pigs.

    Science.gov (United States)

    Lo, Wu-Chia; Chang, Chih-Ming; Liao, Li-Jen; Wang, Chi-Te; Young, Yi-Ho; Chang, Yih-Leong; Cheng, Po-Wen

    2015-01-01

    To date, inadequate study has been devoted to the toxic vestibular effects caused by cisplatin. In addition, no electrophysiological examination has been conducted to assess cisplatin-induced otolith toxicity. The purposes of this study are thus two-fold: 1) to determine whether cervical vestibular-evoked myogenic potentials (VEMPs) and ocular VEMPs are practical electrophysiological methods of testing for cisplatin-induced otolith toxicity and 2) to examine if D-methionine (D-met) pre-injection would protect the otolith organs against cisplatin-induced changes in enzyme activities and/or oxidative status. Guinea pigs were intraperitoneally treated once daily with the following injections for seven consecutive days: sterile 0.9% saline control, cisplatin (5 mg/kg) only, D-met (300 mg/kg) only, or a combination of d-met (300 mg/kg) and cisplatin (5 mg/kg), respectively, with a 30 minute window in between. Each animal underwent the oVEMP and cVEMP tests before and after treatment. The changes in the biochemistry of the otolith organs, including membranous Na(+), K(+)-ATPase and Ca(2+)-ATPase, lipid peroxidation (LPO) levels and nitric oxide (NO) levels, were also evaluated. In the cisplatin-only treated guinea pigs, the mean amplitudes of the oVEMP tests were significantly (potolith dysfunction. D-Met attenuated the reduced ATPase activities and increased oxidative stress induced by cisplatin toxicity in the otolith organs. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Effects of synthetic and naturally occurring flavonoids on Na+, K+-ATPase: Aspects of the structure-activity relationship and action mechanism

    International Nuclear Information System (INIS)

    Hirano, T.; Oka, K.; Akiba, M.

    1989-01-01

    A comparative study was made of the effects of 15 synthetic and naturally occurring flavonoids on the hydrolytic activity of Na + , K + -adenosine triphosphatase (ATPase). Twelve of the flavonoids examined were mono-hydroxy or mono-methoxy derivatives. All inhibited Na + , K + -ATPase from dog kidney cortex when present at concentrations from 40-1000 μM. Flavones possessing cyclohexyl instead of the phenyl group were the most potent with IC 50 at 257-320 μM. Structure-activity relationships were observed among the following mono-substituted flavones as: (i) 2-cyclohexyl-benzopyran-4-one much-gt 2-phenyl-benzopyran-4-one; (ii) 2-cyclohexyl-7-hydroxybenzopyran-4-one > 2-cyclohexyl-6-hydroxy-benzopyran-4-one > 2-cyclohexyl-5-hydroxybenzopyran-4-one. Some flavonoids showing potent inhibitory activity were also examined for ouabain-displacement activity on human erythrocytes. Hardly and of the flavonoids were able to block [ 3 H] ouabain binding to erythrocytes. These results suggest that the mechanism by which flavonoid block Na + , K + -ATPase is not related to the cardiac glycoside-specific binding site(s) of this enzyme

  17. A new member of a family of ATPases is essential for assembly of mitochondrial respiratory chain and ATP synthetase complexes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tzagoloff, A; Yue, J; Jang, J; Paul, M F

    1994-10-21

    Respiration-defective pet mutants of Saccharomyces cerevisiae, assigned to complementation group G25, are grossly deficient in mitochondrial respiratory and ATPase complexes. This phenotype is usually found in strains impaired in mitochondrial protein synthesis. The G25 mutants, however, synthesize all of the proteins encoded b