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

DEFF Research Database (Denmark)

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

2005-01-01

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

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

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.

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.

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)

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.

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

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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

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.

Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments

DEFF Research Database (Denmark)

Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard

2012-01-01

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric...... of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase...... analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any...

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.

Plasma membrane ATPases

DEFF Research Database (Denmark)

Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura

2011-01-01

The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma me...

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Tissue-specific expression of the gene for a putative plasma membrane H(+)-ATPase in a seagrass.

Science.gov (United States)

Fukuhara, T; Pak, J Y; Ohwaki, Y; Tsujimura, H; Nitta, T

1996-01-01

A cDNA clone corresponding to the gene (ZHA1) for a putative plasma membrane H(+)-ATPase of a seagrass (Zostera marina L.) was isolated and sequenced. Comparison of the amino acid predicted sequence from the nucleotide sequence of ZHA1 with those encoded by known genes for plasma membrane H(+)-ATPases from other plants indicated that ZHA1 is most similar to the gene (PMA4) for a plasma membrane H(+)-ATPase in a tobacco (84.4%). Northern hybridization indicated that ZHA1 was strongly expressed in mature leaves, which are exposed to seawater and have the ability of tolerate salinity; ZHA1 was weakly expressed in immature leaves, which are protected from seawater by tightly enveloping sheaths and are sensitive to salinity. In mature leaves, in situ hybridization revealed that ZHA1 was expressed specifically in epidermal cells, the plasma membranes of which were highly invaginated and morphologically similar to those of typical transfer cells. Therefore, the differentiation of the transfer cell-like structures, accompanied by the high-level expression of ZHA1, in the epidermal cells of mature leaves in particular may be important for the excretion of salt by these cells. PMID:8587992

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

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

DEFF Research Database (Denmark)

Palmgren, Michael Gjedde; Buch-Pedersen, Morten Jeppe

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

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

Science.gov (United States)

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

2018-01-01

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

Relationship between changes of plasma endothelin (ET) level, ATPase activity of erythrocyte membrane and development of nephropathy in patients with pregnancy induced hypertension

International Nuclear Information System (INIS)

Qin Lin; Lu Beiyi

2008-01-01

Objective: To investigate the possible role played by alteration of plasma ET levels and activities of Na + - K + -APT ase and Ca 2+ -Mg 2+ -ATPase of erythrocyte membrane in patients with nephropathy pregnancy induced hypertension. Methods: The concentrations of plasma ET was detected with RIA and erythrocyte membrane ATPase activities were determined with Reilni method in 32 pregnant women with PIH complicated with nephropathy and 70 women with PIH but no nephropathy and 35 normal pregnant women as controls. Results: The plasma ET levels in patients with PHI (both with and without nephropathy) were significantly higher than those in normal preganat women (P + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase levels were significantly de- creased (P + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase activity of erythrocyte membrane. (authors)

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

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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.

A Loss in the Plasma Membrane ATPase Activity and Its Recovery Coincides with Incipient Freeze-Thaw Injury and Postthaw Recovery in Onion Bulb Scale Tissue 1

Science.gov (United States)

Arora, Rajeev; Palta, Jiwan P.

1991-01-01

Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase. PMID:16668063

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)

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,...

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...

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

Science.gov (United States)

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

2018-01-01

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

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

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.

[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.

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

Do 14-3-3 proteins and plasma membrane H+-ATPases interact in the barley epidermis in response to the barley powdery mildew fungus?

DEFF Research Database (Denmark)

Finnie, C.; Andersen, C.H.; Borch, J.

2002-01-01

14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon...... inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHA1), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus......, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H...

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.

Identification of antifungal H+-ATPase inhibitors with effect on the plasma membrane potential

DEFF Research Database (Denmark)

Kjellerup, Lasse; Gordon, Sandra; Cohrt, Karen O'Hanlon

2017-01-01

to depolarize the membrane and inhibit extracellular acidification in intact fungal cells, concomitant with a significant increase in intracellular ATP levels. Collectively, we suggest these effects may be a common feature for Pma1 inhibitors. Additionally, the work uncovered a dual mechanism for the previously......The plasma membrane H(+)-ATPase (Pma1) is an essential fungal protein and a proposed target for new antifungal medications. A small-molecule library containing ∼191,000 commercially available compounds was screened for inhibition of S. cerevisiae plasma membranes containing Pma1. The overall hit...... identified cationic peptide BM2, revealing fungal membrane disruption in addition to Pma1 inhibition. The methods presented here provide a solid platform for the evaluation of Pma1-specific inhibitors in a drug development setting. The present inhibitors could serve as a starting point for the development...

Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

National Research Council Canada - National Science Library

VanHouten, Joshua N

2008-01-01

The plasma membrane calcium ATPase isoform 2 (PMCA2) is highly expressed on the apical membrane of mammary epithelial cells during lactation, and is the predominant pump responsible for calcium transport into milk...

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

DEFF Research Database (Denmark)

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

2007-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

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

Directory of Open Access Journals (Sweden)

Kazi Md Kamrul Huda

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

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

MODULATION OF H+-ATPASE ACTIVITY BY FUSICOCCIN IN PLASMA-MEMBRANE VESICLES FROM OAT (AVENA-SATIVA L) ROOTS - A COMPARISON OF MODULATION BY FUSICOCCIN, TRYPSIN, AND LYSOPHOSPHATIDYLCHOLINE

NARCIS (Netherlands)

LANFERMEIJER, FC; PRINS, HBA

The fungal phytotoxin fusicoccin affects various transport processes in the plasma membrane of plant cells. The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) seems to be the primary target of fusicoccin action. The kinetics of the stimulation of the PM H+-ATPase by fusicoccin was studied in PM

[Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations].

Science.gov (United States)

Rybchenko, Zh I; Palladina, T O

2011-01-01

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.

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.

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.

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

Science.gov (United States)

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

2016-01-01

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

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

DEFF Research Database (Denmark)

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

2009-01-01

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

Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H+-ATPase1

Science.gov (United States)

Olivari, Claudio; Albumi, Cristina; Pugliarello, Maria Chiara; De Michelis, Maria Ida

2000-01-01

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H+-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H+-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H+-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H+-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H+-ATPase. PMID:10677439

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...

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

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

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

Science.gov (United States)

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

2016-01-01

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

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

Science.gov (United States)

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

2015-07-01

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

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

Directory of Open Access Journals (Sweden)

Antônio Jesus Dorighetto Cogo

2018-02-01

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

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.

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.

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

Science.gov (United States)

Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

2017-05-01

The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

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

DEFF Research Database (Denmark)

Justesen, Bo Højen

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

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.

Crystal structure of the plasma membrane proton pump

DEFF Research Database (Denmark)

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

2007-01-01

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

Rapid regulation of the plasma membrane H+-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa

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Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey

2015-01-01

Background and Aims The activity of H+-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na+ exclusion via Na+/H+ exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H+-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. Methods The kinetics of salt-induced net H+, Na+ and K+ fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Key Results Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (−144 ± 3·3, −138 ± 5·4 and −128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H+ efflux, Na+ efflux and K+ retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H+ efflux was most pronounced in the root elongation zone. In contrast, H+-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Conclusions Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant’s ability to rapidly upregulate plasma membrane H+-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative

Rapid regulation of the plasma membrane H⁺-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa.

Science.gov (United States)

Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey

2015-02-01

The activity of H(+)-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na(+) exclusion via Na(+)/H(+) exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H(+)-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. The kinetics of salt-induced net H(+), Na(+) and K(+) fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (-144 ± 3·3, -138 ± 5·4 and -128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H(+) efflux, Na(+) efflux and K(+) retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H(+) efflux was most pronounced in the root elongation zone. In contrast, H(+)-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant's ability to rapidly upregulate plasma membrane H(+)-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative membrane potential values and to

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

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Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

High-resolution screening combined with HPLC–HRMS–SPE–NMR for identification of fungal plasma membrane H+-ATPase inhibitors from plants

DEFF Research Database (Denmark)

Kongstad, Kenneth; Wubshet, Sileshi Gizachew; Johannesen, Ane

2014-01-01

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H+-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi...

Isolation and characterization of plasma membranes from guinea pig ileum

International Nuclear Information System (INIS)

Anon.

1986-01-01

A plasma membrane fraction from guinea pig ileum has been isolated by extraction of a crude microsomal fraction with a low ionic strength buffer containing ATP and Ca 2+ . The extracted microsomes were subjected to sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membranes were substantially free from contamination with contractile proteins, mitochondria and sarco-plasmic reticulum. The plasma membrane vesicles were enriched 30-to-40-fold in Na + -K + -ATPase and 5'-nucleotidase activities. The plasma membrane vesicles accumulated Ca 2+ in the presence of ATP. The addition of Ca 2+ ionophore A23187 to vesicles loaded with Ca 2+ in the presence of ATP removed Ca 2+ completely from the vesicles in one minute. The Km values for the Ca 2+ -dependent phosphorylated intermediates of Ca 2+ -Mg 2+ -ATPase and Ca 2+ uptake were approximately 0.8 μM indicating that the phosphorylated intermediates represent phosphorylation of Ca 2+ pump ATPase. The 3 H-nitrendipine binding to plasma membranes was characterized by high affinity with Kd of 185 pM and B/sub max/ 1280 fmol/mg protein. The plasma membrane vesicles prepared by these procedures can prove useful for the study of ion transport

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

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Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

2013-08-01

Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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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.

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

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.

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.

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

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

2010-04-01

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

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....

Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

International Nuclear Information System (INIS)

Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

2010-01-01

Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+-ATPase by combining X-ray crystallography and electron cryomicroscopy

NARCIS (Netherlands)

Ottmann, C.; Marco, S.; Jaspert, N.; Marcon, C.; Schauer, N.; Weyand, M.; Vandermeeren, C.; Duby, G.; Boutry, M.; Wittinghofer, A.; Rigaud, J.-L.; Oecking, C.

2007-01-01

Regulatory 14-3-3 proteins activate the plant plasma membrane H+-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

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Leilismara Sousa

Full Text Available Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1, iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5% than in women and was associated with an increase (446% in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS and an increase (327% in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132% in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

The effect of ultraviolet radiation on wheat root vesicles enriched in plasma membrane

International Nuclear Information System (INIS)

Wright, L.A. Jr.; Murphy, T.M.; Travis, R.L.

1981-01-01

The irradiation of plant cells with UV radiation (254 nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K + -stimulated ATPase was very sensitive to UV radiation (100% inhibition with 2 ). ATPase activity measured in the absence of K + and K + -stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m 2 . (author)

Effect of saline stress on plasma membrane structure and function of barley roots

International Nuclear Information System (INIS)

Rahmani, F. H.

2000-01-01

Barely (Hordeum vulgare L. c v. Black Local) plants were grown hydroponic ally under different saline stresses (50, 100, 150 And 200 mm NaCI. The adverse effect of each saline stress on the structure and function of root cells plasma membrane was studied in terms of root surface ATPase activation by NaCI in the reaction mixture. Was 0, 50, 100. 150 and 200mM. ATPase activity was found to be increased gradually at certain concentrations of NaCI. For control and 50mM stressed plants, the increase in root surface ATPase activity was started at 150mM NaCI. For 100mM stressed plants it was started at 100mM NaCI. For 150 and 200mM stressed plants it was stated at 50mM NaCI Results indicated that the adverse effect of the growth medium saline stresses on the integrity of the plasma membrane was started at 100mM saline stress. Accordingly the role of plasma membrane bound ATPase in active ion transport was disturbed at 100mM saline stress and may be impaired at 150 and 200mM saline stresses. It was suggested that the lipid environment of the plasma membrane surrounding ATPase was modified by the saline stresses 100-200mM. (author). 38 refs., 2 figs., 2 tabs

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...

[Spatial structure of the calixarene-aminophosphonic acids is important for their inhibition of the Na+,K(+)-ATPase activity in plasma membrane of smooth muscle cells].

Science.gov (United States)

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

2010-01-01

It was found that calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) could effectively reduce Na+,K(+)-ATPase activity of the myometrium cell plasmatic membranes (the value of the apparent constant of inhibition I0.5 was 33 +/- 4 nM) while it practically did not influence the "basal" Mg2(+)-ATPase activity of the same membrane. In comparative experiments, we have shown that 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 activities of Na+,K(+)-ATPase and Mg(2+)-ATPase over a wide range of concentrations. Hence, the influence of calixarene C-107 on Na+,K(+)-ATPase activity was caused by the joint action of two aminophosphonic substituents on the upper rim of the calixarene bowl. The isomer of calixarene C-107--calixarene C-160 (5,11-diamino(2-pyridyl)methylphosphono-17,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) also did not influence the Na+,K(+)-ATPase and Mg(2+)-ATPase activities of plasmatic membrane of myometrium cells. We carried out molecular modeling of calixarenes C-107 and C-160 and showed differences in interatomic distance between aminophosphonic substituents of mentioned calixarenes. We came to the conclusion that spatial structure of calixarene C-107, namely localization of two aminophosphonic substituents in 5,17 position of the upper rim of this calixarene, is crucial for inhibition of Na+,K(+)-ATPase activity. Using laser correlation spectroscopy it was found that the 100 microM solution of calixarene C-107 and 2.5% DMSO had microparticles with size range from 100 nm to 10 microm. Plasma membrane vesicles had average hydrodynamic diameter 401 +/- 17 nm, but after interaction of these vesicles with calixarene C-107 we have registered the creation of

Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

Science.gov (United States)

Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

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.

Regulation of alpha1 Na/K-ATPase expression by cholesterol.

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Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

2011-04-29

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.

LEGO-Inspired Drug Design: Unveiling a Class of Benzo[d]thiazoles Containing a 3,4-Dihydroxyphenyl Moiety as Plasma Membrane H+ -ATPase Inhibitors.

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Tung, Truong-Thanh; Dao, Trong T; Junyent, Marta G; Palmgren, Michael; Günther-Pomorski, Thomas; Fuglsang, Anja T; Christensen, Søren B; Nielsen, John

2018-01-08

The fungal plasma membrane H + -ATPase (Pma1p) is a potential target for the discovery of new antifungal agents. Surprisingly, no structure-activity relationship studies for small molecules targeting Pma1p have been reported. Herein, we disclose a LEGO-inspired fragment assembly strategy for the design, synthesis, and discovery of benzo[d]thiazoles containing a 3,4-dihydroxyphenyl moiety as potential Pma1p inhibitors. A series of 2-(benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones was found to inhibit Pma1p, with the most potent IC 50 value of 8 μm in an in vitro plasma membrane H + -ATPase assay. These compounds were also found to strongly inhibit the action of proton pumping when Pma1p was reconstituted into liposomes. 1-(3,4-Dihydroxyphenyl)-2-((6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)ethan-1-one (compound 38) showed inhibitory activities on the growth of Candida albicans and Saccharomyces cerevisiae, which could be correlated and substantiated with the ability to inhibit Pma1p in vitro. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

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Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

2012-01-01

The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

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

Evaluation of plasma membrane calcium/calmodulin-dependent ATPase isoform 4 as a potential target for fertility control.

Science.gov (United States)

Cartwright, Elizabeth J; Neyses, Ludwig

2010-01-01

The array of contraceptives currently available is clearly inadequate and does not meet consumer demands since it is estimated that up to a quarter of all pregnancies worldwide are unintended. There is, therefore, an overwhelming global need to develop new effective, safe, ideally non-hormonal contraceptives for both male and female use. The contraceptive field, unlike other areas such as cancer, has a dearth of new targets. We have addressed this issue and propose that isoform 4 of the plasma membrane calcium ATPase is a potentially exciting novel target for fertility control. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function in the majority of cells is to extrude calcium to the extracellular milieu. Two isoforms of this gene family, PMCA1 and PMCA4, are expressed in spermatozoa, with PMCA4 being the predominant isoform. Although this gene is ubiquitously expressed, its function is highly tissue-specific. Genetic deletion of PMCA4, in PMCA4 knockout mice, led to 100% infertility specifically in the male mutant mice due to a selective defect in sperm motility. It is important to note that the gene deletion did not affect normal mating characteristics in these mice. This phenotype was mimicked in wild-type sperm treated with the non-specific PMCA inhibitor 5-(and 6-) carboxyeosin diacetate succinimidyl ester; a proof-of-principle that inhibition of PMCA4 has potential importance in the control of fertility. This review outlines the potential for PMCA4 to be a novel target for fertility control by acting to inhibit sperm motility. It will outline the characteristics that make this target drugable and will describe methodologies to identify and validate novel inhibitors of this target.

Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

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Askari Amir

2010-05-01

Full Text Available Abstract Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020 successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME, can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

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

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

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.

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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.

[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.

Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

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Reisner, P D; Brandt, P C; Vanaman, T C

1997-01-01

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

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Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

2004-07-01

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

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.

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

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.

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...

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

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

2010-11-09

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

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase.

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Duan, Wenjuan; Zhou, Juefei; Li, Wei; Zhou, Teng; Chen, Qianqian; Yang, Fuyu; Wei, Taotao

2013-04-01

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

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.

Radionuclide assay of membrane Na+, K+-ATPase activity of peserved red blood cells

International Nuclear Information System (INIS)

Trusov, V.V.; Zelenin, A.A.; Marizin, S.A.

1986-01-01

The radionuclide tests were used to investigate the influence of varying blood preservatives on erythrocylic membrane Na + , K + -ATPase activity in samples of whole blood and packed red blood cells from normal donors prepared by standard methods. The tests were performed before and after seven days of preservation under standard conditions. It was found that blood preservations lowered membrane Na + , K + -ATPase activity: its minimum reduction was recorded with citroglucopnosphate, while glugicir induced a significant drop in Na + , K + -ATPase activity of preserved red blood cells regardless of the type of the blood transfusion solution. The assay of membrane Na + , K + -ATPase activity of preserved red blood cells with the use of 86 Rb could be recommended as an evaluation test for preserved blood and its components

Response of membrane-bound ATPase of Micrococcus luteus to heat and ultraviolet light

International Nuclear Information System (INIS)

Volotovskij, J.; Risi, S.; Dose, K.

1976-01-01

It is shown that the properties of ATPase (EC 3.6.1.3) of Micrococcus luteus depend only to some extent on the state of the membrane to which it is attached. Its interaction with the membrane appears to be largely controlled by polar forces. It is shown, however, that the UV-sensitivity of the membrane-bound ATPase is also significantly influenced by the state of membrane lipids. (orig.) [de

Response of membrane-bound ATPase of Micrococcus luteus to heat and ultraviolet light

Energy Technology Data Exchange (ETDEWEB)

Volotovskii, J; Risi, S; Dose, K [Mainz Univ. (F.R. Germany). Inst. fuer Biochemie

1976-03-01

It is shown that the properties of ATPase (EC 3.6.1.3) of Micrococcus luteus depend only to some extent on the state of the membrane to which it is attached. Its interaction with the membrane appears to be largely controlled by polar forces. It is shown, however, that the UV-sensitivity of the membrane-bound ATPase is also significantly influenced by the state of membrane lipids.

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

Plasma Membrane Ca2+-ATPase 4 in Murine Epididymis: Secretion of Splice Variants in the Luminal Fluid and a Role in Sperm Maturation1

OpenAIRE

Patel, Ramkrishna; Al-Dossary, Amal A.; Stabley, Deborah L.; Barone, Carol; Galileo, Deni S.; Strehler, Emanuel E.; Martin-DeLeon, Patricia A.

2013-01-01

Plasma membrane Ca2+-ATPase isoform 4 (PMCA4) is the primary Ca2+ efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca2+/CaM-dependent serine kinase (CASK) in regulating Ca2+ homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we sho...

Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

Science.gov (United States)

Spalding, E. P.; Cosgrove, D. J.

1992-01-01

A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

NARCIS (Netherlands)

Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

2002-01-01

The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

Active calcium transport in plasma membrane vesicles from developing cotyledons of common bean

International Nuclear Information System (INIS)

Huang Jianzhong; Chen Ziyuan

1995-01-01

Plasma membrane vesicles were prepared from the developing cotyledons of common bean (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes. The putative plasma membrane fraction was minimally contaminated by membranes other than plasma membrane and hence was of high purity. It exhibited a Ca 2+ -dependent ATPase activity, which was inhibited by 1 μmol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATP-dependent 45 Ca 2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6 μmol/L CaM and 20 μmol/L IAA but inhibited by 2 μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca 2+ in mediating phytohormones activity is discussed

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

Science.gov (United States)

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Study on the changes in the levels of membrane-bound ATPases ...

African Journals Online (AJOL)

An attempt has been made to determine the deleterious effects of λ cyhalothrin- induced in fresh water tilapia (Oreochromis mossambicus) with respect to changes in the activities of membrane-bound ATPases (Na+/K+, Mg+ and Ca2+ ATPase) and mineral status ...

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

Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase*

Science.gov (United States)

Morton, Michael J.; Farr, Glen A.; Hull, Michael; Capendeguy, Oihana; Horisberger, Jean-Daniel; Caplan, Michael J.

2010-01-01

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys54 in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys54 α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit. PMID:20801885

Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

Science.gov (United States)

Gatto, C; Lutsenko, S; Shin, J M; Sachs, G; Kaplan, J H

1999-05-14

The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

DEFF Research Database (Denmark)

Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg

2010-01-01

Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for p...... group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin...

Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase

OpenAIRE

Andrews, Rachel E.; Galileo, Deni S.; Martin-DeLeon, Patricia A.

2015-01-01

Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca2+ efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca2+, and that these fertility-modulating proteins are present...

The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity.

Science.gov (United States)

Zhao, Jian; Barkla, Bronwyn J; Marshall, Joy; Pittman, Jon K; Hirschi, Kendal D

2008-02-01

Perturbing CAX1, an Arabidopsis vacuolar H+/Ca2+ antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among cax1 and cax3 including an increased sensitivity to both abscisic acid (ABA) and sugar during germination, and an increased tolerance to ethylene during early seedling development. We have also identified phenotypes unique to cax3, namely salt, lithium and low pH sensitivity. We used biochemical measurements to ascribe these cax3 sensitivities to a reduction in vacuolar H+/Ca2+ transport during salt stress and decreased plasma membrane H+-ATPase activity. These findings catalog an array of CAX phenotypes and assign a specific role for CAX3 in response to salt tolerance.

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...

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.

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.

Stimulating effect of low doses of ionizing radiation on the activity of chicken liver and spleen plasma membrane Ca+2 ATPase during different periods of development

International Nuclear Information System (INIS)

Islamov, T.M.

1994-01-01

Effect of pre incubative irradiation of chickens on the activity of chicken liver and spleen plasma membrane Ca +2 -ATPase in 13, 15, 17 day embryos and 1, 5, 10, 20, 30, 40, 50, 60 day chickens has been studied. Low doses of radiation are discovered to stimulate liver and spleen enzyme activity. On the basis of data obtained it is suggested that in the cells of radiosensitive and radio resistive organs molecular mechanisms of stimulating effect of low doses are similar. (author). 10 refs.; 1 fig.; 2 tabs

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

DEFF Research Database (Denmark)

Lopez Marques, Rosa Laura

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

Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 Impairs Pollen Fitness and Alters the Pollen Tube Plasma Membrane

Directory of Open Access Journals (Sweden)

Stephen C McDowell

2015-04-01

Full Text Available Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7 results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions.

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

Science.gov (United States)

Bernhem, Kristoffer; Blom, Hans; Brismar, Hjalmar

2018-01-01

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

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.

[Physiological prion and activity of plasma membrane Na+,K(+)- and Ca(2+)-ATPase in the medulla oblongata cells of rats of different ages].

Science.gov (United States)

Kushkevych, M V; Vlizlo, V V; Martyn, Iu V

2013-01-01

Based on the results of immunohistochemical analysis of the rat medulla tissue the localization of physiological prion has been established. Specifically, in rats aged one month they are placed in the gray matter near the bodies of neurons and mikrohliocytes and in animals of six and thirty months--in olive kernel core and upward path bodies. Physiological prion is localized along the nerve processes and is absent in the neuron bodies. In the medulla oblongata of animals aged six months its amount is the highest compared to animals of other age. The activity of plasma membrane ATPases in this tissue decreases with age, the content of sodium and calcium ions increases, while that of potassium is almost unchanged.

Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: identification of the proteins involved.

Science.gov (United States)

Sreeja, S; Thampan, Raghava Varman

2004-04-01

An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.

Physiological prion and activity of plasma membrane Na(+,K(+- and Ca(2+-ATPase in the medulla oblongata of rats of different ages

Directory of Open Access Journals (Sweden)

M. V. Kushkevych

2013-04-01

Full Text Available Based on the results of immunohistochemical analysis of the rat medulla tissue the localization of physiological prion has been established. Specifically, in rats aged one month they are placed in the gray matter near the bodies of neurons and mikrohliocytes and in animals of six and thirty months – in olive kernel core and upward path bodies. Physiological prion is localized along the nerve processes and is absent in the neuron bodies­. In the medulla oblongata of animals aged six months its amount is the highest compared to animals of other age. The activity of plasma membrane ATPases in this tissue decreases with age, the content of sodium and calcium ions increases, while that of potassium is almost unchanged.

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.

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.

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

Science.gov (United States)

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

Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats.

Science.gov (United States)

Al-Numair, Khalid S; Veeramani, Chinnadurai; Alsaif, Mohammed A; Chandramohan, Govindasamy

2015-01-01

Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities. The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40 mg/kg body weight (BW)). Kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) was administered orally once daily for 45 d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined. In our study, diabetic rats had significantly (p kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) for a period of 45 d resulted in significant (p kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol's action mechanism.

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

Science.gov (United States)

Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

2002-01-01

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

Receptor kinase-mediated control of primary active proton pumping at the plasma membrane

DEFF Research Database (Denmark)

Fuglsang, Anja Thoe; Kristensen, Astrid; Cuin, Tracey A.

2014-01-01

Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro...... and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast...... heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2...

Changes in plasma membrane state of thymocytes during spontaneous and radiation-induced leukemogenesis

International Nuclear Information System (INIS)

Gonta-Grabiec, K.

1984-01-01

Changes in plasma membrane properties characteristic for malignant cells were reviewed. Investigations of spontaneous (in AKR mice) and radiation-induced (in C57Bl) leukemogenesis were carried out; changes in properties of Na + , K + ATPase and alkaline phosphatase were characterized. On the basis of the results reported a pre-leukemic stage was distinguished, corresponding to the following features at the cellular level: increase in activity of alkaline phosphatase; decrease in relative activity of Na + , K + ATPase; decrease in efficiency of the Na + K + pump; decrease in cAMP content. 473 refs. (author)

Density gradient localization of vanadate- and NO-3-sensitive ATPase from sterile cultures of Spirodela polyrrhiza (L. Schleiden

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Józef Buczek

2014-01-01

Full Text Available The present work deals with the separation and some characteristics of ATPase activities bound with plant membanes prepared from sterile cultures of Spirodela polyrrhiza. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density and sensitivity to several inhibitors. The results showed that N0-3-sensitive ATPase activity associated with the tonoplast was localized at a sucrose density between 1.095-1.117 g•cm-3. The vanadate-sensitive ATPase activity bound with the plasma membrane showed a density between 1.127-1.151 g•cm-3. Both ATPases were insensitive to azide and oligomycin and were separable from markers for mitochondria.

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

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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.

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

Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket.

NARCIS (Netherlands)

Koenderink, J.B.; Geibel, S.; Grabsch, E.; Pont, J.J.H.H.M. de; Bamberg, E.; Friedrich, T.

2003-01-01

Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp,

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

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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

Calcium pumps of plasma membrane and cell interior

DEFF Research Database (Denmark)

Strehler, Emanuel E; Treiman, Marek

2004-01-01

Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco....../endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue......- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping...

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

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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.

Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

International Nuclear Information System (INIS)

Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

1986-01-01

The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 2+ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

Regulation of α1 Na/K-ATPase Expression by Cholesterol*

OpenAIRE

Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

2011-01-01

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol tr...

Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

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Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

2013-04-05

Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

Evolution of plant P-type ATPases

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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.

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

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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.

Calcium fluxes across the plasma membrane of Commelina communis L. assayed in a cell-free system

International Nuclear Information System (INIS)

Siebers, B.; Graef, P.; Weiler, E.W.

1990-01-01

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous two-phase partitioning was loaded with 45 Ca 2+ through the action of the plasma membrane Ca 2+ -ATPase. Results suggest the presence of a Ca 2+ channel in the plasma membrane of C. communis. The channel is obtained in a Ca 2+ -inactivated state after preparation and Ca 2+ -loading of the vesicles. The inactivation is removed by TFP [trifluoperazine] or W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], presumably due to the Ca 2+ -mobilizing effect of these compounds. The activated Ca 2+ channel is La 3+ sensitive and, in the cell, would allow for passage of Ca 2+ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed

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

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

2013-11-01

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

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

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

1998-01-01

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

Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

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Farr, Glen A.; Hull, Michael; Stoops, Emily H.; Bateson, Rosalie; Caplan, Michael J.

2015-01-01

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking. PMID:26424804

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

Science.gov (United States)

Menzikov, Sergey A

2017-02-07

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

Preparation of rat gastric heavy and light microsomal membranes enriched in (H+-K+)-ATPase using 2H2O and Percoll gradients

International Nuclear Information System (INIS)

Im, W.B.; Davis, J.P.; Blakeman, D.P.

1985-01-01

Gastric heavy microsomal membranes highly enriched in (H + -K + )-ATPase were obtained from cimetidine- or carbachol-treated rats through 2 H 2 O and Percoll gradient centrifugations. Both the resting (cimetidine-treated) and the stimulated (carbachol-treated) heavy membranes which presumably represent the apical membrane of gastric parietal cells were enriched with the polypeptides of 81,000 and 45,000 besides that of 93,000 representing (H + -K + )-ATPase. No apparent differences could be detected between the resting and the stimulated heavy membranes in their polypeptide profiles or their specific activity of (H + -K + )-ATPase. Nevertheless, the level of 86 RbCl uptake was greater in the stimulated than the resting heavy microsomal membrane vesicles. The light gastric microsomes which abound in intracellular tubulovesicles containing reserve (H + -K + )-ATPase as isolated from cimetidine-treated rats were similarly purified with respect to (H + -K + )-ATPase. The purified light gastric membranes were largely devoid of the polypeptides of 81,000 and 45,000 found in the heavy gastric membranes. These observations further support the current hypothesis that secretagogues bring about changes in the environment of (H + -K + )-ATPase and induce KCl permeability in the apical membrane of the parietal cells, although at present the authors have been unable to identify the polypeptide(s) responsible for the KCl pathway

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

NARCIS (Netherlands)

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

2015-01-01

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

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.

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

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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.

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

International Nuclear Information System (INIS)

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

2004-01-01

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

Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.

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Kumar, Vineet; Prasad, Bhagwat; Patilea, Gabriela; Gupta, Anshul; Salphati, Laurent; Evers, Raymond; Hop, Cornelis E C A; Unadkat, Jashvant D

2015-02-01

To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

Science.gov (United States)

Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

2011-10-01

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

α3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish

DEFF Research Database (Denmark)

Doğanli, Canan; Beck, Hans Christian; Ribera, Angeles B

2013-01-01

Na(+)/K(+)-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na(+)/K(+)-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement ...

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

Science.gov (United States)

Nguyen, Khoa; Garcia, Alvaro; Sani, Marc-Antoine; Diaz, Dil; Dubey, Vikas; Clayton, Daniel; Dal Poggetto, Giovanni; Cornelius, Flemming; Payne, Richard J; Separovic, Frances; Khandelia, Himanshu; Clarke, Ronald J

2018-06-01

The Na + ,K + -ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na + and K + electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31 P and 2 H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Raman Spectroscopy of Conformational Changes in Membrane-Bound Sodium Potassium ATPase

DEFF Research Database (Denmark)

Helix Nielsen, Claus; Abdali, Salim; Lundbæk, Jens August

2007-01-01

In this investigation we assess the potential of Raman spectroscopy as a tool for probing conformational changes in membrane-spanning proteins — in this case, the sodium potassium adenosine triphosphatase (Na+,K+-ATPase). Spectral analysis of protein-lipid complexes is complicated by the presence...

Proteomic screen for multiprotein complexes in synaptic plasma membrane from rat hippocampus by blue native gel electrophoresis and tandem mass spectrometry.

Science.gov (United States)

Li, Xuanwen; Xie, Chunliang; Jin, Qihui; Liu, Mingjun; He, Quanyuan; Cao, Rui; Lin, Yong; Li, Jianglin; Li, Yan; Chen, Ping; Liang, Songping

2009-07-01

Neuronal synapses are specialized sites for information exchange between neurons. Many diseases, such as addiction and mood disorders, likely result from altered expression of synaptic proteins, or altered formation of synaptic complexes involved in neurotransmission or neuroplasticity. A detailed description of native multiprotein complexes in synaptic plasma membranes (PM) is therefore essential for understanding biological mechanisms and disease processes. For the first time in this study, two-dimensional Blue Native/SDS-PAGE electrophoresis, combined with tandem mass spectrometry, was used to screen multiprotein complexes in synaptic plasma membranes from rat hippocampus. As a result, 514 unique proteins were identified, of which 36% were integral membrane proteins. In addition, 19 potentially novel and known heterooligomeric multiprotein complexes were found, such as the SNARE and ATPase complexes. A potentially novel protein complex, involving syntaxin, synapsin I and Na+/K+ ATPase alpha-1, was further confirmed by co-immunoprecipitation and immunofluorescence staining. As demonstrated here, Blue Native-PAGE is a powerful tool for the separation of hydrophobic membrane proteins. The combination of Blue Native-PAGE and mass spectrometry could systematically identify multiprotein complexes.

The α₂Na⁺/K⁺-ATPase is critical for skeletal and heart muscle function in zebrafish

DEFF Research Database (Denmark)

Doganli, Canan; Kjaer-Sørensen, Kasper; Knoeckel, Christopher

2012-01-01

The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase α-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish α2Na+/K+-ATPase associ...

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.

Ist2 in the yeast cortical endoplasmic reticulum promotes trafficking of the amino acid transporter Bap2 to the plasma membrane.

Directory of Open Access Journals (Sweden)

Wendelin Wolf

Full Text Available The equipment of the plasma membrane in Saccharomyces cerevisiae with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H(+ gradient across the plasma membrane generated by the H(+-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of ist2Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.

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.

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…

Atrial Na,K-ATPase increase and potassium dysregulation accentuate the risk of postoperative atrial fibrillation

DEFF Research Database (Denmark)

Tran, Cao Thach; Schmidt, Thomas Andersen; Christensen, John Brochorst

2009-01-01

BACKGROUND: Postoperative atrial fibrillation is a common complication to cardiac surgery. Na,K-ATPase is of major importance for the resting membrane potential and action potential. The purpose of the present study was to evaluate the importance of Na,K-ATPase concentrations in human atrial...... biopsies and plasma potassium concentrations for the development of atrial fibrillation. METHODS: Atrial myocardial biopsies were obtained from 67 patients undergoing open chest cardiac surgery. Na,K-ATPase was quantified using vanadate-facilitated 3H-ouabain binding. Plasma potassium concentration....../g wet weight (n = 33), p = 0.03]. Also with multivariable analysis, 3H-ouabain-binding site concentration was significantly associated with the development of atrial fibrillation. High increase in plasma potassium concentration during the perioperative period and surgery was associated...

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

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.

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

Science.gov (United States)

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

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.

[Effects of desulfurization waste on calcium distribution, Ca(2+)-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress].

Science.gov (United States)

Mao, Gui-Lian; Xu, Xing; Zeng, Jin; Yue, Zi-Hui; Yang, Shu-Juan

2012-02-01

To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca(2+)-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaSO4 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca(2+)-ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2 production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaSO4.

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

OpenAIRE

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-01-01

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analog...

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

DEFF Research Database (Denmark)

Jiang, Qiucen; Garcia, Alvaro; Han, Minwoo

2017-01-01

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

H+ V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes Aegypti

Science.gov (United States)

Brush Border Membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs ) contain an H+ V-ATPase (V), a Na+/H+ antiporter, NHA1 (A) and a Na+-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles...

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

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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.

Analysis of Protein-Membrane Interactions

DEFF Research Database (Denmark)

Kemmer, Gerdi Christine

Cellular membranes are complex structures, consisting of hundreds of different lipids and proteins. These membranes act as barriers between distinct environments, constituting hot spots for many essential functions of the cell, including signaling, energy conversion, and transport. These functions....... Discovered interactions were then probed on the level of the membrane using liposome-based assays. In the second part, a transmembrane protein was investigated. Assays to probe activity of the plasma membrane ATPase (Arabidopsis thaliana H+ -ATPase isoform 2 (AHA2)) in single liposomes using both giant...... are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...

Identification of lysophosphatidylcholine, γ-stearoyl (LPCD) as an endogenous Na+, K+-ATPase inhibitor in volume-expanded hog plasma

International Nuclear Information System (INIS)

Tamura, M.; Inagami, T.

1986-01-01

We have shown that the Na + , K + -ATPase inhibitory activities in the plasma of volume-expanded hog consist of multiple components. One group of the major inhibitory activities induced by intravascular saline infusion was identified as unsaturated free fatty acids. The present study was undertaken to determine the identity of the remaining Na + , K + -ATPase inhibitory activity in the plasma of volume-expanded hogs. Three peaks with ouabain displacing activity (ODA) were separated by HPLC on a reversed phase octadecyl column. The slowest eluting material which showed good solubility in water and recognizable optical absorbance at 214 nm was purified further by three additional steps of reverse phase HPLC. FAB mass spectrometry and 1 H NMR spectroscopy identified this substance as lysophosphatidylcholine, γ-stearoyl. Both purified and synthetic LPCS showed dose-dependent inhibition of Na + , K + -ATPase and displacement of [ 3 H] ouabain from the ATPase. Lysophosphatidylcholines containing either palmitoyl or myristoyl groups also exhibited the Na + , K + -ATPase inhibitory activity and the ODA. The ODA in the LPCS containing fraction increased during the saline infusion. These results indicate that LPCS is an endogenous Na + , K + -ATPase inhibitor which is induced by the expansion of plasma volume

Ninth International Workshop on Plant Membrane Biology

Energy Technology Data Exchange (ETDEWEB)

1993-12-31

This report is a compilation of abstracts from papers which were discussed at a workshop on plant membrane biology. Topics include: plasma membrane ATP-ases; plant-environment interactions, membrane receptors; signal transduction; ion channel physiology; biophysics and molecular biology; vaculor H+ pumps; sugar carriers; membrane transport; and cellular structure and function.

Characterization of cadmium plasma membrane transport in gills of a mangrove crab Ucides cordatus

International Nuclear Information System (INIS)

Ortega, P.; Custódio, M.R.; Zanotto, F.P.

2014-01-01

Highlights: • Cd 2+ gill cell transport, a non-essential toxic metal, was characterized in a hypo-hyper-regulating mangrove crab Ucides cordatus. • Cd 2+ enter gill cells through Ca 2+ channels and is dependent of intracellular Ca 2+ levels. • Route of entry in gill cells also involves a Cd 2+ /Ca 2+ (2Na) exchanger. • Cd transport depends on Na + /K + -ATPase and gill cell electrochemical gradient. • Vanadate inhibits gill Cd 2+ transport and ouabain increase gill Cd 2+ transport. - Abstract: Membrane pathway for intracellular cadmium (Cd 2+ ) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd 2+ transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd 2+ concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd 2+ transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl 2 and different inhibitors. Addition of extracellular calcium (Ca 2+ ) to the cells affected little the fluorescence of FluoZin, confirming that Cd 2+ was the main ion increasing intracellular fluorescence. Ca 2+ channels blockers (nimodipine and verapamil) decreased Cd 2+ influx as well as vanadate, a Ca 2+ -ATPase blocker. Chelating intracellular Ca 2+ (BAPTA) decreased Cd 2+ influx in gill cells, while increasing intracellular Ca 2+ (caffeine) augmented Cd influx. Cd 2+ and ATP added at different temporal conditions were not effective at increasing intracellular Cd 2+ accumulation. Ouabain (Na + /K + -ATPase inhibitor) increased Cd 2+ influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd 2+ influx, a non-essential metal, through the gill cell plasma membrane of crabs are suggested

Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H+-ATPase activity and abundance

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Nikolic Miroslav

2012-05-01

Full Text Available Abstract Background The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level. Results The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction. However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1 and NRT2 transporter (CsNRT2 in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process. Conclusions Obtained results provide for the first time evidence that a saturable

Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling

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Morrill Gene A

2013-01-01

Full Text Available Abstract Background Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. Results [125I]Insulin binding (Kd = 54 ± 6 nM at the oocyte plasma membrane activates membrane serine protease(s, followed by the loss of low affinity ouabain binding sites, with a concomitant 3–4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na+/Ca2+ exchange, increased endocytosis, decreased Na+ conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi. Hyperpolarization is largely due to Na+-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na+/glucose cotransport. The Na+ sym- and antiporter systems are driven by the Na+ free energy gradient generated by Na+/K+-ATPase. Shifts in α and/or β Na+-pump subunits to caveolar (lipid raft membrane regions may activate Na/K-ATPase and contribute to the Na+ free energy gradient and the increase in both Na+/glucose co-transport and pHi. Conclusions Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na+ channels and mobilizes fully functional Na+-pumps, generating a Na+ free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

Effect of washing on the plasma membrane and on stress reactions of cultured rose cells

International Nuclear Information System (INIS)

Qian, Y.C.; Nguyen, T.; Murphy, T.M.

1993-01-01

Cultured cells of Rosa damascena have been used as a model for studies of responses of plant cells to various stresses, including UV radiation, protein-synthesis inhibitors, and elicitors from pathogens. Many of the responses involve reactions at the plasma membrane: efflux of K + , changes in the acid balance between cytoplasm and external medium, synthesis of H 2 O 2 , and inhibition of ferricyanide reduction. In previous studies, the cells have typically been washed with a solution of low ionic strength. We now show that this washing procedure results in changes in the protein composition of the plasma membrane, in the labeling of the proteins in the plasma membrane, and in the specific activity of ATPase in purified plasma membrane vesicles. Also, compared to the unwashed cells, the washed cells show less net K + efflux after UV-C and Phytophthora elicitor treatments; more synthesis of H 2 O 2 after UV-C and a pattern of accumulation of H 2 O 2 after elicitor treatment that shows a delayed but higher peak; and more inhibition of ferricyanide reduction after UV-C, but not after elicitor treatment. The results suggest that washing has differential effects on the mechanisms by which cultured plant cells perceive or respond to two stresses, UV-C and elicitor

The involvement of altered vesicle transport in redistribution of Ca2+, Mg2+-ATPase in cholestatic rat liver

NARCIS (Netherlands)

Song, J. Y.; van Noorden, C. J.; Frederiks, W. M.

1998-01-01

Vectorial sorting of plasma membrane protein-containing vesicles is essential for the establishment and maintenance of cell polarity. In the present study, the involvement of altered vesicle transport in the redistribution of membrane-bound Ca2+, Mg2+-ATPase resulting from cholestasis was

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.

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

NARCIS (Netherlands)

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

2005-01-01

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

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.

Growth inhibitory action of ebselen on fluconazole-resistant Candida albicans: role of the plasma membrane H+-ATPase.

Science.gov (United States)

Billack, Blase; Santoro, Michelle; Lau-Cam, Cesar

2009-06-01

PMA1 is a yeast gene that codes for the plasma membrane H(+)-ATPase, a protein commonly referred to as Pma1p. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a synthetic selenium-containing compound that has recently been shown to display antimicrobial activity owing to its ability to inhibit Pma1p. Ebselen is able to block the activity of Pma1p not only in opportunistic pathogens such as Cryptococcus neoformans and Candida albicans but also in nonpathogenic yeasts such as Saccharomyces cerevisiae. A series of in vitro studies aimed at evaluating the antifungal activity of ebselen were performed. At low concentrations (ebselen was fungistatic against three strains of S. cerevisiae (IC(50) approximately 3 microM) and one fluconazole-resistant strain of C. albicans (IC(50) approximately 6 microM), and at a high concentration (30 microM) it was fungicidal against C. albicans. Moreover, ebselen was found to inhibit medium acidification by the fluconazole-resistant strain of C. albicans in a concentration-dependent manner. In comparison to currently used antifungal agents represented by azole (itraconazole, ketoconazole, fluconazole) and polyene (amphotericin B) compounds, ebselen was at least 10-fold more potent than fluconazole but less active than the other compounds tested. The present results suggest that the growth inhibitory activity of ebselen toward fluconazole-resistant yeast cells is due, at least in part, to inhibition of Pma1p. Ebselen may also serve as a useful agent in the treatment of infections caused by fluconazole-resistant fungi.

Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+-ATPase Activity and K+/Na+ Homeostasis in Sweet Potato.

Science.gov (United States)

Yu, Yicheng; Wang, Aimin; Li, Xiang; Kou, Meng; Wang, Wenjun; Chen, Xianyang; Xu, Tao; Zhu, Mingku; Ma, Daifu; Li, Zongyun; Sun, Jian

2018-01-01

Melatonin (MT) is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [ Ipomoea batatas (L.) Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K + /Na + homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K + /Na + homeostasis in sweet potato seedlings as indicated by the low reduced K + content in tissues and low accumulation of Na + content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K + efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM) H + -ATPase activity and intracellular adenosine triphosphate (ATP) level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG) accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA) β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H + -ATPase activity, and K + /Na + homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H + -ATPase activity and K + /Na + homeostasis in sweet potato.

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

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Summer R. Hayek

2014-01-01

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

Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

Science.gov (United States)

Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

2016-08-01

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

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.

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

DEFF Research Database (Denmark)

Ekberg, Kira; Palmgren, Michael; Veierskov, Bjarke

2010-01-01

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

Involvement of H(+)-ATPase and carbonic anhydrase in inorganic carbon uptake for endosymbiont photosynthesis.

Science.gov (United States)

Furla, P; Allemand, D; Orsenigo, M N

2000-04-01

Symbiotic cnidarians absorb inorganic carbon from seawater to supply intracellular dinoflagellates with CO(2) for their photosynthesis. To determine the mechanism of inorganic carbon transport by animal cells, we used plasma membrane vesicles prepared from ectodermal cells isolated from tentacles of the sea anemone, Anemonia viridis. H(14)CO(-)(3) uptake in the presence of an outward NaCl gradient or inward H(+) gradient, showed no evidence for a Cl(-)- or H(+)- driven HCO(-)(3) transport. H(14)CO(-)(3) and (36)Cl(-) uptakes were stimulated by a positive inside-membrane diffusion potential, suggesting the presence of HCO(-)(3) and Cl(-) conductances. A carbonic anhydrase (CA) activity was measured on plasma membrane (4%) and in the cytoplasm of the ectodermal cells (96%) and was sensitive to acetazolamide (IC(50) = 20 nM) and ethoxyzolamide (IC(50) = 2.5 nM). A strong DIDS-sensitive H(+)-ATPase activity was observed (IC(50) = 14 microM). This activity was also highly sensitive to vanadate and allyl isothiocyanate, two inhibitors of P-type H(+)-ATPases. Present data suggest that HCO(-)(3) absorption by ectodermal cells is carried out by H(+) secretion by H(+)-ATPase, resulting in the formation of carbonic acid in the surrounding seawater, which is quickly dehydrated into CO(2) by a membrane-bound CA. CO(2) then diffuses passively into the cell where it is hydrated in HCO(-)(3) by a cytosolic CA.

Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+–ATPase Activity and K+/Na+ Homeostasis in Sweet Potato

Directory of Open Access Journals (Sweden)

Yicheng Yu

2018-02-01

Full Text Available Melatonin (MT is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [Ipomoea batatas (L. Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K+/Na+ homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K+/Na+ homeostasis in sweet potato seedlings as indicated by the low reduced K+ content in tissues and low accumulation of Na+ content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K+ efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM H+–ATPase activity and intracellular adenosine triphosphate (ATP level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H+–ATPase activity, and K+/Na+ homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H+–ATPase activity and K+/Na+ homeostasis in sweet potato.

Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

Science.gov (United States)

Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

2010-02-01

By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H(+)-ATPase, and one of which is induced by mechanical stress.

Science.gov (United States)

Oufattole, M; Arango, M; Boutry, M

2000-04-01

To analyze in detail the multigene family encoding the plasma-membrane H(+)-ATPase (pma) in Nicotiana plumbaginifolia Viv., five new pma genes (pma 5-9) were isolated. Three of these (pma 6, 8, 9) were fully characterized and classified into new and independent subfamilies. Their cell-type expression was followed by the beta-glucuronidase (gusA) reporter-gene method. While the pma8-gusA transgene was not expressed in transgenic tobacco, expression of the two other transgenes (pma6- and pma9-gusA) was found to be restricted to particular cell types. In the vegetative tissues, pma6-gusA expression was limited to the head cells of the leaf short trichomes, involved in secretion, and to the cortical parenchyma of the young nodes where the developing leaves and axillary flowering stalks join the stem. In the latter tissues, gene expression was enhanced by mechanical stress, suggesting that H(+)-ATPase might be involved in the strength of the tissues and their resistance to mechanical trauma. The pma9-gusA transgene was mainly expressed in the apical meristem of adventitious roots and axillary buds as well as in the phloem tissues of the stem, in which expression depended on the developmental stage. In flowers, pma9-gusA expression was limited to the mature pollen grains and the young fertilized ovules, while that of pma6-gusA was identified in most of the organs. Reverse transcription-polymerase chain reaction of leaf and stem RNA confirmed the expression of pma 6 and 9, while pma8 was found to be expressed in both organs at a lower level. In conclusion, although pma 6 and 9 had a more restricted expression pattern than the previously characterized pma genes, they were nevertheless expressed in cell types in which H(+)-ATPase had not been previously detected.

Isolation of Highly Purified Fractions of Plasma Membrane and Tonoplast from the Same Homogenate of Soybean Hypocotyls by Free-Flow Electrophoresis 1

Science.gov (United States)

Sandelius, Anna Stina; Penel, Claude; Auderset, Guy; Brightman, Andrew; Millard, Merle; Morré, D. James

1986-01-01

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K+-stimulated, vanadate-inhibited Mg2+ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 9 PMID:16664771

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: Presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

Energy Technology Data Exchange (ETDEWEB)

Marxer, A.; Stieger, B.; Quaroni, A.; Kashgarian, M.; Hauri, H.P. (Univ. of Basel (Switzerland))

1989-09-01

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

Liver plasma membranes: an effective method to analyze membrane proteome.

Science.gov (United States)

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

F2α-isoprostane, Na+-K+ ATPase and membrane fluidity of placental syncytiotrophoblast cell in preeclamptic women with vitamin E supplementation

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Franciscus D. Suyatna

2012-11-01

Full Text Available Background: The aim of our study was to analyze F2α-isoprostane level, Na+-K+ ATPase activity and placental syncytiotrophoblast cell membrane fluidity in preeclamptic women who received vitamin E supplementation.Methods: The study was conducted between September 2003 and February 2005 at Budi Kemuliaan Maternity Hospital, Central Jakarta. Samples were 6 preeclamptic women with vitamin E supplementation, 6 preeclamptic women without vitamin E supplementation and 6 normal pregnant women. The dose of vitamin E was 200 mg daily. F2α-isoprostane was measured with ELISA reader at λ of 450 nm. Cell membrane fluidity was measured by comparing the molar ratio of total cholesterol and cell membrane phospholipid concentration. The cholesterol was measured by Modular C800 using Roche reagent. Phospholipid was measured by Shimadzu RF5301PC spectrofluorometer (excitation 267 nm, emission 307 nm. Na+-K+ ATPase activity was inhibited by ouabain. Pi production was measured with Fiske and Subbarow method using spectrophotometer at λ of 660 nm. Data was analyzed using F test with one-way ANOVA.Results: Vitamin E supplementation in preeclamptic women decreased the oxidative stress, indicated by significantly lower level of F2α-isoprostane compared to those without vitamin E (26.72 ± 11.21 vs 41.85 ± 7.09 ng/mL, respectively, p = 0.017. Membrane fluidity in syncytiotrophoblast cell of preeclampsia with vitamin E group was maintained at 0.39 ± 0.08 while in those without vitamin E was 0.53 ± 0.14 (p = 0.04. Na+-K+ ATPase activity in syncytiotrophoblast cell membrane was not affected by vitamin E (p = 0.915.Conclusion: Vitamin E supplementation in preeclamptic women decreases F2α-isoprostane level and maintains cell membrane fluidity of syncytiotrophoblast cells; however, it does not increase Na+-K+ ATPase enzyme activity. (Med J Indones. 2012;21:225-9Keywords: F2α-isoprostane, membrane fluidity, Na+-K+ ATPase, preeclampsia, vitamin E

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

DEFF Research Database (Denmark)

Galuska, Dana; Kotova, Olga; Barres, Romain

2009-01-01

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

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

DEFF Research Database (Denmark)

Veshaguri, Salome

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

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.

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.

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

Science.gov (United States)

Clausen, Michael V; Hilbers, Florian; Poulsen, Hanne

2017-01-01

The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

Characterization of cadmium plasma membrane transport in gills of a mangrove crab Ucides cordatus

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Ortega, P.; Custódio, M.R. [Instituto de Biociências, Departamento de Fisiologia, Universidade de São Paulo, Rua do Matão, Travessa 14, #101, São Paulo 05508-900, SP (Brazil); Zanotto, F.P., E-mail: fzanotto@usp.br [Instituto de Biociências, Departamento de Fisiologia, Universidade de São Paulo, Rua do Matão, Travessa 14, #101, São Paulo 05508-900, SP (Brazil); Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, São Paulo 04044-020 (Brazil)

2014-12-15

Highlights: • Cd{sup 2+} gill cell transport, a non-essential toxic metal, was characterized in a hypo-hyper-regulating mangrove crab Ucides cordatus. • Cd{sup 2+} enter gill cells through Ca{sup 2+} channels and is dependent of intracellular Ca{sup 2+} levels. • Route of entry in gill cells also involves a Cd{sup 2+}/Ca{sup 2+} (2Na) exchanger. • Cd transport depends on Na{sup +}/K{sup +}-ATPase and gill cell electrochemical gradient. • Vanadate inhibits gill Cd{sup 2+} transport and ouabain increase gill Cd{sup 2+} transport. - Abstract: Membrane pathway for intracellular cadmium (Cd{sup 2+}) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd{sup 2+} transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd{sup 2+} concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd{sup 2+} transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl{sub 2} and different inhibitors. Addition of extracellular calcium (Ca{sup 2+}) to the cells affected little the fluorescence of FluoZin, confirming that Cd{sup 2+} was the main ion increasing intracellular fluorescence. Ca{sup 2+} channels blockers (nimodipine and verapamil) decreased Cd{sup 2+} influx as well as vanadate, a Ca{sup 2+}-ATPase blocker. Chelating intracellular Ca{sup 2+} (BAPTA) decreased Cd{sup 2+} influx in gill cells, while increasing intracellular Ca{sup 2+} (caffeine) augmented Cd influx. Cd{sup 2+} and ATP added at different temporal conditions were not effective at increasing intracellular Cd{sup 2+} accumulation. Ouabain (Na{sup +}/K{sup +}-ATPase inhibitor) increased Cd{sup 2+} influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd{sup 2+} influx, a non-essential metal, through the

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

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

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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.

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

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Igor I. Krivoi

2016-05-01

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

Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

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Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

2014-09-05

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Chromium-induced membrane damage: protective role of ascorbic acid.

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Dey, S K; Nayak, P; Roy, S

2001-07-01

Importance of chromium as environmental toxicant is largely due to impact on the body to produce cellular toxicity. The impact of chromium and their supplementation with ascorbic acid was studied on plasma membrane of liver and kidney in male Wistar rats (80-100 g body weight). It has been observed that the intoxication with chromium (i.p.) at the dose of 0.8 mg/100 g body weight per day for a period of 28 days causes significant increase in the level of cholesterol and decrease in the level of phospholipid of both liver and kidney. The alkaline phosphatase, total ATPase and Na(+)-K(+)-ATPase activities were significantly decreased in both liver and kidney after chromium treatment, except total ATPase activity of kidney. It is suggested that chromium exposure at the present dose and duration induce for the alterations of structure and function of both liver and kidney plasma membrane. Ascorbic acid (i.p. at the dose of 0.5 mg/100 g body weight per day for period of 28 days) supplementation can reduce these structural changes in the plasma membrane of liver and kidney. But the functional changes can not be completely replenished by the ascorbic acid supplementation in response to chromium exposure. So it is also suggested that ascorbic acid (nutritional antioxidant) is useful free radical scavenger to restrain the chromium-induced membrane damage.

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

Membrane order in the plasma membrane and endocytic recycling compartment.

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Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

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.

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

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Michael V. Clausen

2017-06-01

Full Text Available The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

Decrease of Na, K-ATPase Electrogenic Contribution and Resting Membrane Potential of Rat Soleus after 3 Days of Hindlimb Unloading

Science.gov (United States)

Krivoi, I. I.; Kravtsova, V. V.; Drabkina, T. M.; Prokofiev, A. V.; Nikolsky, E. E.; Shenkman, B. S.

2008-06-01

The Na,K-ATPase activity is critically important for excitability, electrogenesis and contractility of skeletal muscle expressing ? and ? isoforms of the enzyme [6, 9]. It is well known that disuse induced by hindlimb unloading (HU) leads to progressive atrophy of skeletal muscle; the muscle undergoes a number of dramatic remodeling events. In particular, changes in ion channel expression in response to muscle unweighting were observed [1, 8]. Decrease of resting membrane potential (RMP), electrogenic contribution of Na,K-ATPase and membrane resistance during 7-28 days of HU was shown [8, 10]. The intrinsic mechanisms involved in the process have not been revealed until present. At the same time, the understanding of these mechanisms could be crucial for the disclosing the mechanisms underlying the resting Ca2+ accumulation in the cytoplasm of the unloaded muscle [3, 7]. In the present study, the effect of early (3 days) HU-induced disuse of slow-twitch soleus muscle on membrane electrogenesis as well as on electrogenic contribution of Na,K-ATPase isoforms was investigated.

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase

International Nuclear Information System (INIS)

Pestov, Nikolay B.; Dmitriev, Ruslan I.; Kostina, Maria B.; Korneenko, Tatyana V.; Shakhparonov, Mikhail I.; Modyanov, Nikolai N.

2012-01-01

Highlights: ► Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. ► ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. ► Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. ► Subcellular localization of SPCA2 may depend on tissue type. ► In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis

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Vitart, V.; Baxter, I.; Doerner, P.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2001-01-01

The plasma membrane of plant cells is energized by an electrochemical gradient produced by P-type H+-ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter-GUS reporter assays. A disruption of this pump (aha4-1) was identified as a T-DNA insertion in the middle of the gene (after VFP(574)). Truncated aha4-1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild-type plants. Plants homozygous for aha4-1 (-/-) show a subtle reduction in root and shoot growth compared with wild-type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mM NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4-5-fold. Interestingly, the aha4-1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild-type copies of AHA4. These results are consistent with the hypothesis that aha4-1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.

Preparation of wheat root plasma membrane vesicles and effect of water stress on 45Ca2+ transport activity

International Nuclear Information System (INIS)

Lu Jinyin; Gao Junfeng; Cao Cuiling

1998-01-01

The wheat roots plasma membrane (PM) vesicles were obtained by sucrose gradient centrifugation. The experiment results shows that the wheat roots of Zhengyin No.1 PM H + -ATPase latent activity was 24%, and PM inside-out vesicle (IOV) accounts for 76%. With -1.0 MPa stress of 24h, PM Ca 2+ -ATPase activity of both orientation wheat roots were increased. Under normal water condition and PEG stress, 62% and 53% of the enzyme activity was inhibited respectively by EGTA, radioactive calcium-45 transport amount was 22.09 nmol/mg pro and 4.17 nmol/mg pro. respectively with PM-IOV.PEG stress results in a decrease of 45 Ca 2+ transport amount of wheat roots PM-IOV by 81%

In vitro assessment of the growth and plasma membrane H+ -ATPase inhibitory activity of ebselen and structurally related selenium- and sulfur-containing compounds in Candida albicans.

Science.gov (United States)

Orie, Natalie N; Warren, Andrew R; Basaric, Jovana; Lau-Cam, Cesar; Piętka-Ottlik, Magdalena; Młochowski, Jacek; Billack, Blase

2017-06-01

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H + -ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)-resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC 50  ∼ 18 μM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC 50  ∼ 14 μM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU-resistant fungi. © 2017 Wiley Periodicals, Inc.

NMR spectroscopic studies of membrane-bound biological systems

International Nuclear Information System (INIS)

Hohlweg, W.

2013-01-01

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

Biogenesis of plasma membrane cholesterol

International Nuclear Information System (INIS)

Lange, Y.

1986-01-01

A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from [ 3 H] acetate moved to the plasma membrane with a half-time of 1 h at 37 0 C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane

Athermal alterations in the structure in the canalicular membrane and ATPase activity induced by thermal levels of microwave radiation

International Nuclear Information System (INIS)

Phelan, A.M.; Neubauer, C.F.; Timm, R.; Neirenberg, J.; Lange, D.G.

1994-01-01

Sprague-Dawley rats (200-250 g) were exposed 30 min/day for 4 days to thermogenic levels (rectal temperature increase of 2.2 degrees C) of microwave radiation [2.45 GHz, 80 mW/cm 2 , continuous-wave mode (CW)] or to a radiant heat source resulting in an equivalent increase in body temperature of 2.2 degrees C. On the fifth day the animals were sacrificed and their livers removed. The canalicular membranes were isolated and evaluated for adenosinetriphosphatase (ATPase) activity, total fatty acid composition and membrane fluidity characteristics. Mg ++ -ATPase activity (V max ) decreased by 48.5% in the group exposed to microwave radiation, with no significant change in the group exposed to radiant heat. The decrease in Mg ++ -ATPase was partially compensated by a concomitant increase in Na + /K + -ATPase activity (170% increase in V max over control) in animals exposed to microwave radiation, while no change occurred in the group exposed to radiant heat. This alteration in ATPase activity in the group exposed to microwave radiation is associated with a large decrease in the ratio of saturated to unsaturated fatty acids. Conversely, the group exposed to radiant heat had an increase in the ratio of saturated to unsaturated fatty acids. The most dramatic changes were found in the levels of arachidonic acid. Finally, the electron paramagnetic resonance (EPR) spin label technique used to measure the fluidity of the canalicular membranes of the animals in the three groups (sham, microwave radiation and radiant heat) indicated that the results were different in the three groups, reflecting the changes found in their fatty acid composition. The physiological response to open-quotes equivalentclose quotes thermal loads in rats is expressed differently for different types of energy sources. Possible mechanisms producing these divergent thermogenic responses are discussed. 34 refs., 3 figs., 2 tabs

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

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca{sup 2+}-ATPase

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Pestov, Nikolay B., E-mail: korn@mail.ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Dmitriev, Ruslan I.; Kostina, Maria B. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Korneenko, Tatyana V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shakhparonov, Mikhail I. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

2012-01-27

Highlights: Black-Right-Pointing-Pointer Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. Black-Right-Pointing-Pointer ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. Black-Right-Pointing-Pointer Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. Black-Right-Pointing-Pointer Subcellular localization of SPCA2 may depend on tissue type. Black-Right-Pointing-Pointer In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2

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

DEFF Research Database (Denmark)

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

1989-01-01

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

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

Science.gov (United States)

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

2015-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Tsong, T.Y.

1990-06-30

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

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

Science.gov (United States)

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

2012-12-01

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

Proteomics and the dynamic plasma membrane

DEFF Research Database (Denmark)

Sprenger, Richard R; Jensen, Ole Nørregaard

2010-01-01

plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required...... for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

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

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.

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.

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

Membrane lipid microenvironment modulates thermodynamic properties of the Na+-K+-ATPase in branchial and intestinal epithelia in euryhaline fish in vivo

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Mario Diaz

2016-12-01

Full Text Available We have analyzed the effects of different native membrane lipid composition on the thermodynamic properties of the Na+-K+-ATPase in different epithelia from the gilthead seabream Sparus aurata. Thermodynamic parameters of activation for the Na+-K+-ATPase, as well as contents of lipid classes and fatty acids from polar lipids were determined for gill epithelia and enterocytes isolated from pyloric caeca, anterior intestine and posterior intestine. Arrhenius analyses of control animals revealed differences in thermal discontinuity values (Td and activation energies determined at both sides of Td between intestinal and gill epithelia. Eyring plots disclosed important differences in enthalpy of activation (H‡ and entropy of activation (S‡ between enterocytes and branchial cells. Induction of n-3 LCPUFA deficiency dramatically altered membrane lipid composition in enterocytes, being the most dramatic changes the increase in 18:1n-9 (oleic acid and the reduction of n-3 LCPUFA (mainly DHA, docosahexaenoic acid. Strikingly, branchial cells were much more resistant to diet-induced lipid alterations than enterocytes, indicating the existence of potent lipostatic mechanisms preserving membrane lipid matrix in gill epithelia. Paralleling lipid alterations, values of Ea1, H‡ and S‡ for the Na+-K+-ATPase were all increased, while Td values vanished, in LCPUFA deficient enterocytes. In turn, Differences in thermodynamic parameters were highly correlated with specific changes in fatty acids, but not with individual lipid classes including cholesterol in vivo. Thus, Td was positively related to 18:1n-9 and negatively to DHA. Td, Ea1 and H‡ were exponentially related to DHA/18:1n-9 ratio. The exponential nature of these relationships highlights the strong impact of subtle changes in the contents of oleic acid and DHA in setting the thermodynamic properties of epithelial Na+-K+-ATPase in vivo. The effects are consistent with physical

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques

International Nuclear Information System (INIS)

Mahajan, S.; Lewis, R.N.; George, R.; Sykes, B.D.; McElhaney, R.N.

1988-01-01

The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22 Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism

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.

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

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.

ATPase activity of erythrocyte membranes and their permeability for the K-ions as influenced by irradiation and serotonin

International Nuclear Information System (INIS)

Zhegnevskaya, V.V.; Vinogradova, M.F.; Polevoj, V.V.

1982-01-01

Na, K-ATPase activity of membranes of erytrocytes after 1 hour of X-ray irradiation of citrate blood of rats (25.8 Kl/kg)-increased, and after irradiation of isolated erytrocytes, placed in the isotonic solution of NaCl did not change. The exflux of K-ions out of irradiated erytrocytes increased equally in both cases. Serotonin (2x10 -4 M), added to the probes 10 minutes before irradiation, decreased the exflux of K + by irradiated erytrocytes, but Na, K-ATPase activity under the influence of amine was without changes

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

Directory of Open Access Journals (Sweden)

Craig A Goodman

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

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.

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.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Plasma membrane Ca2+-ATPase 4 in murine epididymis: secretion of splice variants in the luminal fluid and a role in sperm maturation.

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Patel, Ramkrishna; Al-Dossary, Amal A; Stabley, Deborah L; Barone, Carol; Galileo, Deni S; Strehler, Emanuel E; Martin-DeLeon, Patricia A

2013-07-01

Plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is the primary Ca(2+) efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca(2+)/CaM-dependent serine kinase (CASK) in regulating Ca(2+) homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT-PCR, that both the splice variants of Pmca4 mRNA are expressed in murine testis and throughout the epididymis. Immunofluorescence localized PMCA4a to the apical membrane of the epididymal epithelium, and Western analysis not only confirmed its presence but showed for the first time that PMCA4a and PMCA4b are secreted in the epididymal luminal fluid (ELF), from which epididymosomes containing PMCA4a were isolated. Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5-fold compared to caput sperm (detected by Western blotting) and ~2-fold after incubation in ELF, revealing in vitro uptake and implicating PMCA4a in epididymal sperm maturation. Coimmunoprecipitation using pan-PMCA4 antibodies, revealed that both variants associate with CASK, suggesting their presence in a complex. Because they have different kinetic properties for Ca(2+) transport and different abilities to bind to CASK, our study suggests a mechanism for combining the functional attributes of both PMCA4 variants, leading to heightened efficiency of the pump in the maintenance of Ca(2+) homeostasis, which is crucial for normal motility and male fertility.

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

DEFF Research Database (Denmark)

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

1997-01-01

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

Analysis of tomato plasma membrane H(+)-ATPase gene family suggests a mycorrhiza-mediated regulatory mechanism conserved in diverse plant species.

Science.gov (United States)

Liu, Junli; Liu, Jianjian; Chen, Aiqun; Ji, Minjie; Chen, Jiadong; Yang, Xiaofeng; Gu, Mian; Qu, Hongye; Xu, Guohua

2016-10-01

In plants, the plasma membrane H(+)-ATPase (HA) is considered to play a crucial role in regulating plant growth and respoding to environment stresses. Multiple paralogous genes encoding different isozymes of HA have been identified and characterized in several model plants, while limited information of the HA gene family is available to date for tomato. Here, we describe the molecular and expression features of eight HA-encoding genes (SlHA1-8) from tomato. All these genes are interrupted by multiple introns with conserved positions. SlHA1, 2, and 4 were widely expressed in all tissues, while SlHA5, 6, and 7 were almost only expressed in flowers. SlHA8, the transcripts of which were barely detectable under normal or nutrient-/salt-stress growth conditions, was strongly activated in arbuscular mycorrhizal (AM) fungal-colonized roots. Extreme lack of SlHA8 expression in M161, a mutant defective to AM fungal colonization, provided genetic evidence towards the dependence of its expression on AM symbiosis. A 1521-bp SlHA8 promoter could direct the GUS reporter expression specifically in colonized cells of transgenic tobacco, soybean, and rice mycorrhizal roots. Promoter deletion assay revealed a 223-bp promoter fragment of SlHA8 containing a variant of AM-specific cis-element MYCS (vMYCS) sufficient to confer the AM-induced activity. Targeted deletion of this motif in the corresponding promoter region causes complete abolishment of GUS staining in mycorrhizal roots. Together, these results lend cogent evidence towards the evolutionary conservation of a potential regulatory mechanism mediating the activation of AM-responsive HA genes in diverse mycorrhizal plant species.

Lipid organization of the plasma membrane

NARCIS (Netherlands)

Ingólfsson, Helgi I; Melo, Manuel N; van Eerden, Floris J; Arnarez, Clément; Lopez, Cesar A; Wassenaar, Tsjerk A; Periole, Xavier; de Vries, Alex H; Tieleman, D Peter; Marrink, Siewert J

2014-01-01

The detailed organization of cellular membranes remains rather elusive. Based on large-scale molecular dynamics simulations, we provide a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity. Our plasma membrane model consists of 63 different

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.

The plasma membrane as radiosensitive target

International Nuclear Information System (INIS)

Koeteles, Gy.J.

1986-01-01

Components and conditions rendering the plasma membrane susceptible for ionizing radiation are discussed. The list of reviews and articles pointing to various aspects of radiation effects on membranes is analyzed. Radiation induced alterations of plasma membrane and energy deposition in cellular microstructures are overviewed. The possible role of membrane alterations in the fate of irradiated cell is also discussed. (author)

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

Science.gov (United States)

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

DEFF Research Database (Denmark)

Alexander, R Todd; Beggs, Megan R; Zamani, Reza

2015-01-01

role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestine, while pan-specific Pmca antibodies...... the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In human kidney, a similar pattern of distribution was observed, with highest PMCA4 expression in NCC positive tubules. Electron microscopy demonstrated Pmca4 localization...... in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments, but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing...

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Science.gov (United States)

Ward, M. R.; Grimes, H. D.; Huffaker, R. C.

1989-01-01

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

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...

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

Energy Technology Data Exchange (ETDEWEB)

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

1987-05-19

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

Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.

Science.gov (United States)

Suñé, Guillermo; Sarró, Eduard; Puigmulé, Marta; López-Hellín, Joan; Zufferey, Madeleine; Pertel, Thomas; Luban, Jeremy; Meseguer, Anna

2010-11-10

Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

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.

Inhibition of rat synaptic membrane Na⁺/K⁺-ATPase and ecto-nucleoside triphosphate diphosphohydrolases by 12-tungstosilicic and 12-tungstophosphoric acid.

Science.gov (United States)

Čolović, Mirjana B; Bajuk-Bogdanović, Danica V; Avramović, Nataša S; Holclajtner-Antunović, Ivanka D; Bošnjaković-Pavlović, Nada S; Vasić, Vesna M; Krstić, Danijela Z

2011-12-01

The in vitro influence of Keggin structure polyoxotungstates, 12-tungstosilicic acid, H(4)SiW(12)O(40) (WSiA) and 12-tungstophosphoric acid, H(3)PW(12)O(40) (WPA), and monomer Na(2)WO(4) × 2H(2)O on rat synaptic plasma membrane (SPM) Na(+)/K(+)-ATPase and E-NTPDase activity was studied, whereas the commercial porcine cerebral cortex Na(+)/K(+)-ATPase served as a reference. Dose-dependent Na(+)/K(+)-ATPase inhibition was obtained for all investigated compounds. Calculated IC(50) (10 min) values, in mol/l, for SPM/commercial Na(+)/K(+)-ATPase, were: 3.4 × 10(-6)/4.3 × 10(-6), 2.9 × 10(-6)/3.1 × 10(-6) and 1.3 × 10(-3)/1.5 × 10(-3) for WSiA, WPA and Na(2)WO(4) × 2H(2)O, respectively. In the case of E-NTPDase, increasing concentrations of WSiA and WPA induced its activity reduction, while Na(2)WO(4) × 2H(2)O did not noticeably affect the enzyme activity at all investigated concentrations (up to 1 × 10(-3)mol/l). IC(50) (10 min) values, obtained from the inhibition curves, were (in mol/l): 4.1 × 10(-6) for WSiA and 1.6 × 10(-6) for WPA. Monolacunary Keggin anion was found as the main active molecular species present under physiological conditions (in the enzyme assays, pH 7.4), for the both polyoxotungstates solutions (1 mmol/l), using Fourier transform infrared (FT-IR) and micro-Raman spectroscopy. Additionally, commercial porcine cerebral cortex Na(+)/K(+)-ATPase was exposed to the mixture of Na(2)WO(4) × 2H(2)O and WSiA at different concentrations. Additive inhibition effect was achieved for lower concentrations of Na(2)WO(4) × 2H(2)O/WSiA (≤ 1 × 10(-3)/4 × 10(-6) mol/l), while antagonistic effect was obtained for all higher concentrations of the inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Insulin promotes Rip11 accumulation at the plasma membrane by inhibiting a dynamin- and PI3-kinase-dependent, but Akt-independent, internalisation event.

Science.gov (United States)

Boal, Frédéric; Hodgson, Lorna R; Reed, Sam E; Yarwood, Sophie E; Just, Victoria J; Stephens, David J; McCaffrey, Mary W; Tavaré, Jeremy M

2016-01-01

Rip11 is a Rab11 effector protein that has been shown to be important in controlling the trafficking of several intracellular cargoes, including the fatty acid transporter FAT/CD36, V-ATPase and the glucose transporter GLUT4. We have previously demonstrated that Rip11 translocates to the plasma membrane in response to insulin and here we examine the basis of this regulated phenomenon in more detail. We show that Rip11 rapidly recycles between the cell interior and surface, and that the ability of insulin to increase the appearance of Rip11 at the cell surface involves an inhibition of Rip11 internalisation from the plasma membrane. By contrast the hormone has no effect on the rate of Rip11 translocation towards the plasma membrane. The ability of insulin to inhibit Rip11 internalisation requires dynamin and class I PI3-kinases, but is independent of the activation of the protein kinase Akt; characteristics which are very similar to the mechanism by which insulin inhibits GLUT4 endocytosis. Copyright © 2015. Published by Elsevier Inc.

Crystal structure of Na+, K(+)-ATPase in the Na(+)-bound state.

Science.gov (United States)

Nyblom, Maria; Poulsen, Hanne; Gourdon, Pontus; Reinhard, Linda; Andersson, Magnus; Lindahl, Erik; Fedosova, Natalya; Nissen, Poul

2013-10-04

The Na(+), K(+)-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na(+) and K(+) across the plasma membrane--a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K(+)-bound or ouabain-blocked forms. We present the crystal structure of a Na(+)-bound Na(+), K(+)-ATPase as determined at 4.3 Å resolution. Compared with the K(+)-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na(+) sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na(+) from IIIb through IIIa, followed by exchange of Na(+) for K(+) at sites I and II, is suggested.

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

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

Science.gov (United States)

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

Science.gov (United States)

Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

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

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

Science.gov (United States)

Poëa-Guyon, Sandrine; Ammar, Mohamed Raafet; Erard, Marie; Amar, Muriel; Moreau, Alexandre W; Fossier, Philippe; Gleize, Vincent; Vitale, Nicolas; Morel, Nicolas

2013-10-28

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity.

Science.gov (United States)

Olli, Kristine E; Li, Kun; Galileo, Deni S; Martin-DeLeon, Patricia A

2018-01-01

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca 2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca 2+ efflux pump, PMCA4. This is the major Ca 2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca 2+ ] in capacitated and Ca 2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca 2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca 2+ -dependent manner. In Pmca4 -/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca 2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4 -/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance. © 2017 Wiley Periodicals, Inc.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

Science.gov (United States)

Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A; Hassan, Ali B; Giles, Jennifer

2015-05-22

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Science.gov (United States)

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

2014-01-01

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

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

Science.gov (United States)

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

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

Directory of Open Access Journals (Sweden)

Huiying Zhang

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

Preliminary studies on the concentration of Na+,K(+)-ATPase in skeletal muscle of draught cattle in Mozambique: effect of sex, age and training.

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Veeneklaas, R J; Verkleij, C B; van Schie, B; Harun, M A S; Everts, M E

2002-09-01

The effect of training on the potential for work in draught cattle was assessed by measuring the Na+,K(+)-ATPase in the muscle cell membrane and the elevation in the concentration of K+ in plasma during exercise. Biopsies of the semitendinosus muscle and venous blood samples were taken from the cattle used for draught work in Mozambique. No differences were found in the plasma ion or Na+,K(+)-ATPase concentrations in samples taken from Nguni, Africander and Angoni breeds. There were no significant differences in plasma ions (Na+,K+ and Cl-) or muscle Na+,K(+)-ATPase concentrations between the Angoni males and females, although the males showed an increase in Na+,K(+)-ATPase with age, while the females showed a decrease. The increase in males might be attributed to their higher level of activity in the herds than that of females. After a training period of 15 days, a significant increase in Na+,K(+)-ATPase concentration in semitendinosus muscle was found in Angoni cattle. In females, this was significant after 8 days of training (about 30%); in males after 15 days of training (about 16%). On day 15, there was a reduction in the elevation of plasma K+ during the 2 h of draught work, indicating an increased capacity of the Na+,K+ pumps to maintain the extracellular K+ concentration in working muscles and a possible delay in the moment of fatigue.

Hydrogen superpermeable membrane operation under plasma conditions

International Nuclear Information System (INIS)

Bacal, M.; Bruneteau, A.M.; Livshits, A.I.; Alimov, V.N.; Notkin, M.E.

2003-01-01

The effect of ion bombardment on hydrogen plasma-driven permeation through a superpermeable niobium membrane was investigated. It was found that the increase of membrane temperature and the doping of membrane material with oxygen results in the decrease of ion bombardment effect and in permeability increase. It was demonstrated that membrane decarbonization leads to the formation of a membrane state resistant to sputtering. Possible applications of the membrane resistant to ion bombardment as plasma facing components are considered

Internal hydration of a metal-transporting ATPase is controlled by membrane lateral pressure

Energy Technology Data Exchange (ETDEWEB)

Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Fischermeier, E. [Technische Univ. Dresden (Germany); Pospisil, P. [A.S.C. R., Prague (Czech Republic). J. Heyrovsky Inst. Physical Chemistry; Solioz, M. [Bern Univ. (Switzerland); Sayed, A.; Hof, M.

2017-07-01

The active transport of ions across biological mem branes requires their hydration shell to interact with the interior of membrane proteins. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Using the octahelical transmembrane architecture of the copper-transporting P{sub 1B}-type ATPase from Legionella pneumophila (LpCopA) as a model structure, we have established the site-specific labeling of internal cysteines with a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the protein and in its lipid-embedded state in nano-discs (NDs). Time-dependent fluorescence shifts revealed the site-specific hydration and dipole mobility around the conserved ion-binding motif. The spatial distribution of both features is shaped significantly and independently of each other by membrane lateral pressure.

Internal hydration of a metal-transporting ATPase is controlled by membrane lateral pressure

International Nuclear Information System (INIS)

Fahmy, Karim; Pospisil, P.; Sayed, A.; Hof, M.

2017-01-01

The active transport of ions across biological mem branes requires their hydration shell to interact with the interior of membrane proteins. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Using the octahelical transmembrane architecture of the copper-transporting P_1_B-type ATPase from Legionella pneumophila (LpCopA) as a model structure, we have established the site-specific labeling of internal cysteines with a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the protein and in its lipid-embedded state in nano-discs (NDs). Time-dependent fluorescence shifts revealed the site-specific hydration and dipole mobility around the conserved ion-binding motif. The spatial distribution of both features is shaped significantly and independently of each other by membrane lateral pressure.

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

Energy Technology Data Exchange (ETDEWEB)

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

1985-08-01

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

Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.

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Guillermo Suñé

Full Text Available Cyclophilins (Cyps, the intracellular receptors for Cyclosporine A (CsA, are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1 that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

Effect of therapeutic concentration of lithium on live HEK293 cells, increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane

Czech Academy of Sciences Publication Activity Database

Vošahlíková, Miroslava; Ujčíková, Hana; Chernyavskiy, Oleksandr; Brejchová, Jana; Roubalová, Lenka; Alda, M.; Svoboda, Petr

2017-01-01

Roč. 1861, č. 5 (2017), s. 1099-1112 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GAP207/12/0919; GA ČR(CZ) GA15-16605S; GA ČR(CZ) GA17-07070S Institutional support: RVO:67985823 Keywords : lithium * HEK293 cells * Na+/K+-ATPase * membrane biophysics * proteomic analysis Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient

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Shrivastava, Amulya Nidhi; Redeker, Virginie; Fritz, Nicolas; Pieri, Laura; Almeida, Leandro G; Spolidoro, Maria; Liebmann, Thomas; Bousset, Luc; Renner, Marianne; Léna, Clément; Aperia, Anita; Melki, Ronald; Triller, Antoine

2015-01-01

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC. PMID:26323479

The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

Science.gov (United States)

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-06-17

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions

Czech Academy of Sciences Publication Activity Database

Zahrádka, Jaromír; Sychrová, Hana

2012-01-01

Roč. 12, č. 4 (2012), s. 439-446 ISSN 1567-1356 R&D Projects: GA MŠk(CZ) LC531; GA AV ČR(CZ) IAA500110801 Grant - others:Univerzita Karlova(CZ) 33779266 Institutional research plan: CEZ:AV0Z50110509 Keywords : potassium uptake * potassium efflux * yeast * plasma-membrane potential Subject RIV: EE - Microbiology, Virology Impact factor: 2.462, year: 2012

Iron overload impact on P-ATPases.

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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.

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

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

2010-12-01

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

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

OpenAIRE

Doudová, Lenka

2017-01-01

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

Final Report for DE-FG02-04ER15626: P-type ATPases in Plants – Role of Lipid Flippases in Membrane Biogenesis

Energy Technology Data Exchange (ETDEWEB)

Harper, Jeffrey F. [Univ. of Nevada, Reno, NV (United States)

2015-02-24

The long-range goal of the research is to understand the structure and biological functions of different P-type ATPases (ion pumps) in plant cells, and to use that knowledge to enhance the production of bioenergy from plants, or plant-research inspired technologies. Ptype ATPases include ion pumps that specifically transport H⁺, Ca²+, Zn²⁺, Cu²⁺, K⁺, or Na⁺, as well as at least one unusual subfamily that appears to function as lipid flippases, flipping specific lipids from one side of a membrane bilayer to the other. As a group, P-type ATPases are thought to consume more than 1/3 of the cellular ATP in typical eukaryotic cells. Recent research in the Harper lab focused on understanding the biochemical and biological functions of P-type ATPases that flip lipids. These flippases belong to the P4 subfamily of P-type ATPases. The activity of lipid flippases is thought to induce membrane curvature and/or create an asymmetry in which certain lipid head groups are preferential exposed to one surface or the other. In Arabidopsis thaliana there are 12 members of this family referred to as Aminophospholipid ATPase (ALA) 1 to ALA12. Using genetic knockouts, the Harper lab has established that this unusual subfamily of P-type ATPases are critical for plants to cope with even modest changes in temperature (e.g., down to 15°C, or up to 30°C). In addition, members of one subclade are critical for cell expansion, and loss of function mutants result in severe dwarfism. Other members of this same sub-clade are critical for pollen tube growth, and loss of function mutants are sterile under conditions of hot days and cold nights. While the cellular processes that depend on lipid flippases are still unclear, the genetic analysis of loss of function mutants clearly show they are of fundamental importance to plant growth and response to the environment.

Caveolae as plasma membrane sensors, protectors and organizers.

Science.gov (United States)

Parton, Robert G; del Pozo, Miguel A

2013-02-01

Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

Science.gov (United States)

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

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.

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

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Yasser A Mahmmoud

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

Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

Science.gov (United States)

Bevers, Edouard M; Williamson, Patrick L

2016-04-01

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure. Copyright © 2016 the American Physiological Society.

Expression and Trafficking of the γ Subunit of Na,K-ATPase in Hypertonically Challenged IMCD3 Cells

International Nuclear Information System (INIS)

Pihakaski-Maunsbach, Kaarina; Nonaka, Shoichi; Maunsbach, Arvid B.

2008-01-01

The γ subunit (FXYD2) of Na,K-ATPase is an important regulator of the sodium pump. In this investigation we have analysed the trafficking of γ to the plasma membrane in cultures of inner medullary collecting duct cells (IMCD3) following acute hypertonic challenge and brefeldin A (BFA) treatment. Following hypertonic challenging for 24 hr immunofluorescence labeling revealed initial co-localization of the γ subunit and 58K Golgi protein in the cytoplasm, but no co-localization of α1 and Golgi protein. Exposure of the challenged cells to BFA prevented the subsequent incorporation of γ into the basolateral plasma membrane. The γ subunit instead remained in cytoplasmic vesicles while cell proliferation and cell viability decreased simultaneously. Following removal of BFA from the hypertonic medium the IMCD3 cells recovered with distinct expression of γ in the basolateral membrane. The α1 subunit was only marginally influenced by BFA. The results demonstrate that the γ subunit trafficks to the plasma membrane via the Golgi apparatus, despite the absence of a signal sequence. The results also suggest that the γ and α subunits do not traffic together to the plasma membrane, and that the γ and α subunit have different turnover rates during these experimental conditions

Imbalance of plasma membrane ion leak and pump relationship as a new aetiological basis of certain disease states.

Science.gov (United States)

Ronquist, G; Waldenström, A

2003-12-01

The basis for life is the ability of the cell to maintain ion gradients across biological membranes. Such gradients are created by specific membrane-bound ion pumps [adenosine triphosphatases (ATPases)]. According to physicochemical rules passive forces equilibrate (dissipate) ion gradients. The cholesterol/phospholipid ratio of the membrane and the degree of saturation of phospholipid fatty acids are important factors for membrane molecular order and herewith a determinant of the degree of non-specific membrane leakiness. Other operative principles, i.e. specific ion channels can be opened and closed according to mechanisms that are specific to the cell. Certain compounds called ionophores can be integrated in the plasma membrane and permit specific inorganic ions to pass. Irrespective of which mechanism ions leak across the plasma membrane the homeostasis may be kept by increasing ion pumping (ATPase activity) in an attempt to restore the physiological ion gradient. The energy source for this work seems to be glycolytically derived ATP formation. Thus an increase in ion pumping is reflected by increased ATP hydrolysis and rate of glycolysis. This can be measured as an accumulation of breakdown products of ATP and end-products of anaerobic glycolysis (lactate). In certain disease entities, the balance between ATP formation and ion pumping may be disordered resulting in a decrease in inter alia (i.a.) cellular energy charge, and an increase in lactate formation and catabolites of adenylates. Cardiac syndrome X is proposed to be due to an excessive leakage of potassium ions, leading to electrocardiographic (ECG) changes, abnormal Tl-scintigraphy of the heart and anginal pain (induced by adenosine). Cocksackie B3 infections, a common agent in myocarditis might also induce an ionophore-like effect. Moreover, Alzheimer's disease is characterized by the formation of extracellular amyloid deposits in the brain of patients. Perturbation of cellular membranes by the

Experimental study of membrane pump for plasma devices

International Nuclear Information System (INIS)

Suzuki, Hajime; Ohyabu, Nobuyoshi; Nakamura, Yukio; Sagara, Akio; Motojima, Osamu; Livshits, A.; Notkin, M.; Busnyuk, A.; Komatsu, Kazuyuki

1998-01-01

Recycling control is a key to improve fusion plasma performance. The membrane pump has potential advantages for hydrogen pumping in fusion devices. However, there are unsolved issues for using membrane pump in LHD (Large Helical Device). The first issue is characteristics of the membrane pump under high incident hydrogen atom flux. The second issue is relationship between the surface condition and the pumping efficiency. Impurities from plasma may change the surface condition of the membrane. In order to solve these issues, a membrane pump system was fabricated and installed in a linear plasma device at NIFS (National Institute for Fusion Science). The membrane pump was successfully operated. (author)

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

Plasma membrane disruption: repair, prevention, adaptation

Science.gov (United States)

McNeil, Paul L.; Steinhardt, Richard A.

2003-01-01

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

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

General and specific lipid-protein interactions in Na,K-ATPase.

Science.gov (United States)

Cornelius, F; Habeck, M; Kanai, R; Toyoshima, C; Karlish, S J D

2015-09-01

The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions." Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

The plasma membrane proteome of germinating barley embryos

DEFF Research Database (Denmark)

Hynek, Radovan; Svensson, Birte; Jensen, O.N.

2009-01-01

Cereal seed germination involves a complex coordination between different seed tissues. Plasma membranes must play crucial roles in coordination and execution of germination; however, very little is known about seed plasma membrane proteomes due to limited tissue amounts combined...... with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two-phase partitioning. Reversed-phase chromatography on C-4 resin performed in micro-spin columns with stepwise elution by 2-propanol...... was used to reduce soluble protein contamination and enrich for hydrophobic proteins. Sixty-one proteins in 14 SDS-PAGE bands were identified by LC-MS/MS and database searches. The identifications provide new insight into the plasma membrane functions in seed germination....

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-09-26

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

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

Science.gov (United States)

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

2014-01-01

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

Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

Science.gov (United States)

Carmona-Salazar, Laura; El Hafidi, Mohammed; Gutiérrez-Nájera, Nora; Noyola-Martínez, Liliana; González-Solís, Ariadna; Gavilanes-Ruíz, Marina

2015-01-01

It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

Science.gov (United States)

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Isolation of plasma membrane-associated membranes from rat liver.

Science.gov (United States)

Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

2014-02-01

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

Oxidative stress (glutathionylation and Na,K-ATPase activity in rat skeletal muscle.

Directory of Open Access Journals (Sweden)

Carsten Juel

Full Text Available Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation on the Na,K-ATPase in rat skeletal muscle membranes.Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05 in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0-10 mM increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.

Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

Science.gov (United States)

Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

2015-11-01

Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at

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...

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

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.

Gamma-ray-induced changes in hypodermal mesocarp tissue plasma membrane of pre- and post-storage muskmelon

International Nuclear Information System (INIS)

Lester, G.E.; Whitaker, B.D.

1996-01-01

Gamma irradiation (1.0 kGy) of intact, newly harvested, mature muskmelon fruit (Cucumis melo L. var. reticulatus Naud.) appears to have an immediate deleterious effect, but also a long-term beneficial effect, on the integrity and function of the plasma membrane (PM) of hypodermal mesocarp tissue. The initial consequences of gamma irradiation included an increase in the free sterol:phospholipid ratio, resulting at least in part from deglycosylation of steryl glycosides, a decrease in the spinasterol:7-stigmastenol ratio in each of the PM steryl lipids (free sterols, steryl glycosides, and acylated steryl glycosides), and a decrease in H+-ATPase activity. Irradiation did not increase protein loss, suggesting that the decrease in H+-ATPase activity resulted from either direct inactivation of the enzyme or altered PM ordering caused by the steryl lipid modifications. The long-term beneficial effects of irradiation, observed following 10 days of commercial storage, included greater retention of total PM protein, a diminished decline in total PM phospholipids (PL) and in the PL:protein ratio, and maintenance of greater overall H-ATPase activity (activity was the same as in controls on a per mg protein basis, but there was gt 30% more protein in the PM of stored irradiated fruit). These results indicate that 1 kGy gamma irradiation administered prior to storage slowes the progression of two key parameters of senescence, PM protein loss and PL catabolism

Large-scale analysis of in Vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

DEFF Research Database (Denmark)

Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N

2003-01-01

specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from...... plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage...... of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane...

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

Science.gov (United States)

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

2005-01-01

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

Plant plasma membrane proteomics for improving cold tolerance

Directory of Open Access Journals (Sweden)

Daisuke eTakahashi

2013-04-01

Full Text Available Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation. One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells

NARCIS (Netherlands)

Mays, R.W.; Siemers, K.A.; Fritz, B.A.; Lowe, A.W.; van Meer, G.; Nelson, W.J.

1995-01-01

We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state.

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)

At the border: the plasma membrane-cell wall continuum.

Science.gov (United States)

Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

2015-03-01

Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

OpenAIRE

Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

2006-01-01

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

Beneficial effects of gamma linolenic acid supplementation on nerve conduction velocity, Na+, K+ ATPase activity, and membrane fatty acid composition in sciatic nerve of diabetic rats.

Science.gov (United States)

Coste, T; Pierlovisi, M; Leonardi, J; Dufayet, D; Gerbi, A; Lafont, H; Vague, P; Raccah, D

1999-07-01

Metabolic and vascular abnormalities are implicated in the pathogenesis of diabetic neuropathy. Two principal metabolic defects are altered lipid metabolism resulting from the impairment of delta-6-desaturase, which converts linoleic acid (LA) into gamma linolenic acid (GLA), and reduced nerve Na+, K+ ATPase activity. This reduction may be caused by a lack of incorporation of (n-6) fatty acids in membrane phospholipids. Because this ubiquitous enzyme maintains the membrane electrical potential and allows repolarization, disturbances in its activity can alter the process of nerve conduction velocity (NCV). We studied the effects of supplementation with GLA (260 mg per day) on NCV, fatty acid phospholipid composition, and Na+, K+ ATPase activity in streptozotocin-diabetic rats. Six groups of 10 rats were studied. Two groups served as controls supplemented with GLA or sunflower oil (GLA free). Two groups with different durations of diabetes were studied: 6 weeks with no supplementation and 12 weeks supplemented with sunflower oil. To test the ability of GLA to prevent or reverse the effects of diabetes, two groups of diabetic rats were supplemented with GLA, one group for 12 weeks and one group for 6 weeks, starting 6 weeks after diabetes induction. Diabetes resulted in a 25% decrease in NCV (P < 0.0001), a 45% decrease in Na+, K+ ATPase activity (P < 0.0001), and an abnormal phospholipid fatty acid composition. GLA restored NCV both in the prevention and reversal studies and partially restored Na+, K+ ATPase activity in the preventive treatment group (P < 0.0001). These effects were accompanied by a modification of phospholipid fatty acid composition in nerve membranes. Overall, the results suggest that membrane fatty acid composition plays a direct role in NCV and confirm the beneficial effect of GLA supplementation in diabetic neuropathy.

Light-induced modification of plant plasma membrane ion transport.

Science.gov (United States)

Marten, I; Deeken, R; Hedrich, R; Roelfsema, M R G

2010-09-01

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

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.

Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles.

Science.gov (United States)

Moche, Martin; Stremlau, Stefanie; Hecht, Lars; Göbel, Cornelia; Feussner, Ivo; Stöhr, Christine

2010-01-01

Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10-15 mM nitrate.

Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

Science.gov (United States)

Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

2017-02-01

Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPS B.cep. ) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPS B.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H + -ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Protons and how they are transported by proton pumps

DEFF Research Database (Denmark)

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

2008-01-01

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

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).

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.

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

2015-11-01

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

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...

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

Science.gov (United States)

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Relationship between serum leptin levels, ATPase activity of erythrocyte membrance and development of diabetic nephropathy in patients with DM2

International Nuclear Information System (INIS)

Wang Yuming

2009-01-01

Objective: To study the possible mechanism of development of nephrosis affected by changes of serum leptin levels and alteration of activities of Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase of erythrocyte membrane in patients with type 2 diabetes(DM2). Methods: Serum leptin levels (with RIA) and erythrocyte membrane Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase activitities (with Reinila method) were determined in 40 DM2 patients without nephropathy, 32 DM2 patients with nephropathy and 35 controls. Results Serum leptin levels were significantly higher in the diabetics as a whole than those in controls (P + K + -ATPase and Ca 2+ Mg 2+ -ATPase activities were significantly lower (P<0.01). Among the diabetic patients, the serum leptin levels in patients without nephrosis (P<0.05), but the RBC membrance ATPase activities were significantly lower(P<0.05). Conclusion: Development of type 2 diabetes nephrosis might be correlated with the high serum leptin level and decreased ATPase activities of erythrocite membrane. (authors)

Nanoclustering as a dominant feature of plasma membrane organization

NARCIS (Netherlands)

Garcia-Parajo, M.F.; Cambi, A.; Torreno-Pina, J.A.; Thompson, N.; Jacobson, K.

2014-01-01

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at

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...

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

Science.gov (United States)

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-01-01

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K+·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K+, in marked contrast to previous models. Due to antagonism between ouabain and K+, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity. PMID:19666591

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain.

Science.gov (United States)

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-08-18

The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane

OpenAIRE

1990-01-01

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this c...

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

Directory of Open Access Journals (Sweden)

Anna Bodzęta

2017-08-01

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

Annexins are instrumental for efficient plasma membrane repair in cancer cells.

Science.gov (United States)

Lauritzen, Stine Prehn; Boye, Theresa Louise; Nylandsted, Jesper

2015-09-01

Plasma membrane stress can cause damage to the plasma membrane, both when imposed by the extracellular environment and by enhanced oxidative stress. Cells cope with these injuries by rapidly activating their plasma membrane repair system, which is triggered by Ca(2+) influx at the wound site. The repair system is highly dynamic, depends on both lipid and protein components, and include cytoskeletal reorganization, membrane replacements, and membrane fusion events. Cancer cells experience enhanced membrane stress when navigating through dense extracellular matrix, which increases the frequency of membrane injuries. In addition, increased motility and oxidative stress further increase the risk of plasma membrane lesions. Cancer cells compensate by overexpressing Annexin proteins including Annexin A2 (ANXA2). Annexin family members can facilitate membrane fusion events and wound healing by binding to negatively charged phospholipids in the plasma membrane. Plasma membrane repair in cancer cells depends on ANXA2 protein, which is recruited to the wound site and forms a complex with the Ca(2+)-binding EF-hand protein S100A11. Here they regulate actin accumulation around the wound perimeter, which is required for wound closure. In this review, we will discuss the requirement for Annexins, S100 proteins and actin cytoskeleton in the plasma membrane repair response of cancer cells, which reveals a novel avenue for targeting metastatic cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plasma-polymerized alkaline anion-exchange membrane: Synthesis and structure characterization

International Nuclear Information System (INIS)

Hu Jue; Meng Yuedong; Zhang Chengxu; Fang Shidong

2011-01-01

After-glow discharge plasma polymerization was developed for alkaline anion-exchange membranes synthesis using vinylbenzyl chloride as monomer. X-ray photoelectron spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were used to characterize the chemical structure properties of plasma-polymerized membranes. Ion-exchange capacities of quaternized poly(vinylbenzyl chloride) (QPVBC) membranes were measured to evaluate their capability of hydroxyl ion transport. A mechanism of plasma polymerization using VBC as monomer that accounts for the competitive effects of free radicals polymerization and plasma ablation in the plasma polymerization process was proposed. Our results indicate that plasma discharge power influences the contents of functional groups and the structure of the plasma polymer membranes, which attribute to the coactions of polymerization and ablation. The properties of uniform morphology, good adhesion to the substrate, high thermal stability and satisfying anion conduction level suggest the potential application of QPVBC membrane deposited at discharge power of 20 W in alkaline direct methanol fuel cells.

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

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.

SwissProt search result: AK100483 [KOME

Lifescience Database Archive (English)

Full Text Available AK100483 J023097B04 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 7e-23 ...

SwissProt search result: AK120057 [KOME

Lifescience Database Archive (English)

Full Text Available AK120057 J013000P10 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 1e-88 ...

SwissProt search result: AK120057 [KOME

Lifescience Database Archive (English)

Full Text Available AK120057 J013000P10 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 2e-89 ...

SwissProt search result: AK105082 [KOME

Lifescience Database Archive (English)

Full Text Available AK105082 001-046-C08 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 6e-15 ...

SwissProt search result: AK241795 [KOME

Lifescience Database Archive (English)

Full Text Available AK241795 J065208E19 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 5e-15 ...

SwissProt search result: AK058787 [KOME

Lifescience Database Archive (English)

Full Text Available AK058787 001-002-E05 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-65 ...

SwissProt search result: AK120495 [KOME

Lifescience Database Archive (English)

Full Text Available AK120495 J013120H18 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 1e-142 ...

SwissProt search result: AK070260 [KOME

Lifescience Database Archive (English)