Plasma Membrane ATPase Activity Research Articles

An activation of synaptosomal Na+,K(+)-ATPase by a novel dibenzoxazepine derivative (BY-1949) in the rat brain: its functional role in the neurotransmitter uptake systems.

In search of factors mitigating the final outcome of ischemic and epileptic brain damage, we tested a novel dibenzoxazepine derivative (BY-1949), as the compound has been shown to be effective under these two conditions. First, using rat brain, we assessed whether or not BY-1949 affects the Na+,K(+)-ATPase activity. Although in vitro applications of either BY-1949 or its three major metabolites did not cause any apparent effects, both acute and chronic oral administrations of the compound (10 mg/kg) invariably increased the Na+,K(+)-ATPase activity in the synaptosomal plasma membranes by increasing Vmax values. Second, it was shown by this study that the drug treatment caused marked increases in the uptake of both glutamic acid and gamma-aminobutyric acid into the synaptosomes. These results suggest that the activity against ischemic/epileptic brain damage by BY-1949 is explicable, at least partly, in terms of improvement of ionic derangements across the neural membranes via Na+,K(+)-ATPase activation.

The Phospholipid Platelet-activating Factor Stimulates Proton Extrusion in Cultured Soybean Cells and Protein Phosphorylation and ATPase Activity in Plasma Membranes

Summary Medium acidification by cell cultures from soybean ( Glycine max. L.) was stimulated by the ether phospholipid platelet-activating factor (PAF), 1-O-alkyl-2-acetyl- sn -glycero-3-phosphocholine. Acidification was dependent on the concentration of PAF, on the cell density and on the presence of calcium ions in the medium. Ethyleneglycol-bis(aminoethylether)-N,N,N〲,N〲-tetraacetic acid (EGTA) equaling the calcium concentration reduced the proton extrusion whereas addition of the calcium ionophore A 23187 increased it. Cells in the logarithmic growth phase 3—4 days after inoculation, showed the strongest response to the lipid. In vitro PAF had a stimulatory effect on the ATPase activity in both the tonoplast-and the plasma membrane-enriched fraction. In microsomes isolated from cultured soybean cells PAF stimulated the phosphorylation of a 55 kDa, a 97 kDa and a 120 kDa polypeptide. With an antibody prepared against a cytosolic fragment of the plasma membrane H + -ATPase an immunostaining signal was obtained at 120 kDa, suggesting that the phosphoprotein having the same molecular mass could be the plasma membrane H + -ATPase. After membrane fractionation by a sucrose gradient the 55 kDa and the 97 kDa phosphoproteins were found predominantly in fractions of low density correlating with markers of either tonoplast or endoplasmatic reticulum.

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretes a so-called “suppressor“ in its pycnospore germination fluid. The suppressor blocks the defense responses and induces local susceptibility (accessibility) in pea plants to agents that are not pathogenic in pea. The suppressor nonspecifically inhibits the ATPase activity in plasma membranes prepared from pea, soybean, kidney bean, cowpea and barley plants. However, cytochemical studies by electron microscopy indicate that the suppressor specifically inhibits the ATPase in pea cell membranes, but not in those of four other plant species tested. That is, the specificity of the suppressor appears at the cell and/or tissue level, but is not evident in vitro. Furthermore, the inhibitory effect of the suppressor is temporary because the ATPase activity recovers 9 h after the treatment. A similar effect was observed after inoculation with M. pinodes but not with a nonpathogen of pea, M. ligulicola. The role of the suppressor in host-parasite specificity is discussed.

Normalization of Na+-K+-ATPase Activity in Isolated Membrane Fraction From Sciatic Nerves of Streptozocin-Induced Diabetic Rats by Dietary myo-Inositol Supplementation in Vivo or Protein Kinase C Agonists in Vitro

A myo-inositol-related defect in nerve Na(+)-K(+)-ATPase in experimental diabetes has been invoked in the pathogenesis of diabetic neuropathy, but the mechanism linking altered myo-inositol metabolism and Na(+)-K(+)-ATPase regulation in diabetic nerve is uncertain. Decreased Na(+)-K(+)-ATPase in diabetic rat nerve is normalized by aldose reductase inhibitors or dietary myo-inositol, which preserve normal nerve myo-inositol content in vivo. Decreased Na(+)-K(+)-ATPase in diabetic rabbit nerve is acutely reversed by exposure to protein kinase C agonists in vitro. This study explored the relationship between the myo-inositol-sensitive and protein kinase C-agonist-sensitive Na(+)-K(+)-ATPase defects in diabetic rat nerve. Ouabain-sensitive ATPase activity was measured in an enriched membrane fraction isolated from nondiabetic, streptozocin-induced diabetic, and myo-inositol-supplemented streptozocin-induced diabetic rats before and after the membranes were exposed to protein kinase C agonists in vitro. The decreased ouabain-sensitive ATPase activity in plasma membranes from untreated diabetic rats was increased after exposure to two structurally unrelated protein kinase C agonists; the normal ouabain-sensitive ATPase in plasma membranes from myo-inositol-supplemented diabetic rats was unaffected by protein kinase C agonists. The nonadditivity and implied equivalence of the Na(+)-K(+)-ATPase defect corrected by myo-inositol in vivo and by protein kinase C agonists in vitro are consistent with the postulated existence of a deficient myo-inositol-dependent phospholipid-derived protein kinase C agonist (presumably diacylglycerol) in diabetic nerve that regulates nerve Na(+)-K(+)-ATPase either directly or via a protein kinase C mechanism.

Neomycin Inhibits the Phosphatidylinositol Monophosphate and Phosphatidylinositol Bisphosphate Stimulation of Plasma Membrane ATPase Activity

The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301). In this paper, we show the effect of various concentrations of phosphatidyinositol, PIP, and PIP(2) on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP(2) at concentrations of 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.

Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4-50 mg l-1 (0.03-0.35 mM), pH 4.0] was 1.5-fold higher than that in cells grown in its absence. The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.

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

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.

Age differences in transport ATP-ase activity in plasma membranes of small intestinal enterocytes

Age differences in transport ATP-ase activity in plasma membranes of small intestinal enterocytes

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.

Regulation of nutrient transfer between host and fungus in vesicular-arbuscular mycorrhizas.

Although the overwhelming majority of non-aquatic vascular plants form vesicular-arbuscular (VA) mycorrhizal associations, the extent of colonization of the host root by any given fungal symbiont varies considerably depending on host and environmental factors. Because VA mycorrhizal fungi are obligate biotrophs, transfer of photosynthate from host to fungus may be an important factor in regulating the extent of VA mycorrhizal formation. Host metabolites must cross the plasma membrane before becoming available to the fungus. Several studies on rates of root exudation under various environmental conditions show a strong correlation between rates of root exudation and percent of root length colonized by VA mycorrhizal fungi. However, passive leakage of simple metabolites from roots as the sole means of regulating fungal colonization seems improbable for an obligate biotroph which has not yet been successfully cultured on any artificial medium. So far there has been insufficient investigation of hormone interactions between symbionts, and of the interference by the fungus in host cell wall synthesis, to evaluate the possible role of these factors in controlling growth of VA mycorrhizal fungi. Cytochemical studies of the host-fungus interface suggest modification of host plasma membrane ATPase activity as arbuscules develop, but the function of this altered activity remains unresolved. The presence of a linked P1 -photosynthate exchange mechanism on the host plasma membrane analogous to the P1 -photosynthate translocator known to exist in the outer membrane of chloroplasts remains an uninvestigated possible mechanism for balancing photosynthate demand by the fungus with enhanced P uptake.

Cytochemical localization of plasma membrane ATPase activity in plant cells

The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, β-glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H+-ATPase activity are summarized in relation to previous criticisms of these methods.

Long-term stabilization and crystallization of (Ca 2+ + Mg 2+)-ATPase of detergent-solubilized erythrocyte plasma membrane

Conditions which were optimal for the stabilization of Ca 2+-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Pikula, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277–5286) were also found conductive for preservation of (Ca 2+ + Mg 2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca 2+-dependent activity of (Ca 2+ + Mg 2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca 2+ (10–20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca 2+ + Mg 2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca 2+ + Mg 2+)-ATPase differ from those of Ca 2+-ATPase of detergent-solubilized sarcopiasmic reticulum microsomes.

The role of the plasma membrane atpase in bentazone-sethoxydim antagonism

This study was carried out to gain more information concerning the mechanism by which bentazone reduces the herbicidal activity of sethoxydim. The possible roles of ATP and of the plasma membrane ATPase activity in the mechanism of antagonism were investigated. The first part of the study investigated the effect of exogenous ATP on the uptake of [14C]sethoxydim by wheat leaf segments. Uptake increased with ATP concentration, the magnitude of the increase depending on the time of day when the incubations were started. In the second part of the study plasma membranes from wheat leaves were isolated using two immiscible polymers, polyethylene glygol and dextran, and the activities of their ATPases were measured by monitoring the amount of inorganic phosphate produced. ATPase activity was lower in the presence of 500 μM bentazone and the magnitude of the decrease increased with the concentration of bentazone. Bentazone derivatives that reduce the uptake of sethoxydim but have no effect on photosynthesis also reduced ATPase activity. A mechanism is proposed by which bentazone could alter the uptake of sethoxydim by decreasing the activity of plasma membrane ATPases.

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

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.

Modifications induced by general anesthetics on Na+/K+ ATPase obtained from human placenta

Previously it was demonstrated that thiopental in vivo anesthesia didn't affect the Na+/K(+)-ATPase activity of syncythiotrophoblast plasma membrane, while affecting other enzymatic activity. The aim of the present work was to investigate if this lack of effect of thiopental on the Na+/K+ ATPase activity might be due to its specificity of action on definite membrane proteins or if the binding sites of the anesthetic to this enzyme might be masked within the membrane. Temperature dependence of the Na+/K(+)-ATPase activity and of a spin label paramagnetic maleimide derivative (MSL,2,2,6,6-tetramethylpiperidin-1-oxyl-4-maleimide), which shows a selective binding to the reduced sulfhydryl groups of proteins were investigated. This report shows that a Na+/K(+)-ATPase membranous preparation obtained from placental tissue is strongly inhibited by thiopental.

Rapid light-induced changes in phosphoinositide kinases and H(+)-ATPase in plasma membrane of sunflower hypocotyls.

Irradiation of sunflower (Helianthus annuus L. cv. Russian Mammoth) hypocotyls with white light resulted in a 51% decrease in plasma membrane phosphatidylinositol monophosphate (PIP) kinase activity. As little as 10 s of white light irradiation was sufficient to lower the phosphatidylinositol bisphosphate (PIP2) produced in the in vitro phosphorylation assay. This decrease was not caused by an increase in phospholipase C activity since analysis of the water-soluble products indicated no increase in inositol bisphosphate or inositol trisphosphate. Treatment of the plasma membrane with 200 microM vanadate prior to phosphorylation enhanced the PIP kinase and appeared to overcome the light inhibition. In addition to decreasing the PIP kinase activity, light irradiation resulted in a corresponding decrease in the H(+)-ATPase activity to 53% of the dark control values. The plasma membrane ATPase activity increased approximately 2-fold when PIP or PIP2 was added to the isolated membranes. Thus, effects of external stimuli on the level of plasma membrane PIP or PIP2 could affect plasma membrane ATPase activity directly and thereby provide an alternative mechanism for control of cell growth.

Plasma-membrane ATPase action affects several stress tolerances of Saccharomyces cerevisiae and Schizosaccharomyces pombe as well as the extent and duration of the heat shock response

The pma1.1 mutations of Saccharomyces cerevisiae and Schizosaccharomyces pombe decrease plasma-membrane ATPase activity. This study investigated how they affect different stress tolerances, and the extent and duration of the heat-shock response. pma1.1 mutants exhibited higher resistance to ethanol and osmotic stress, but lower tolerance to ultraviolet damage, as compared to wild-type cells. pma1.1 mutations also increased tolerance of the lethal temperature of 48 °C in cells in which no heat-shock response had been induced. However, after induction of a heat-shock response and elevated thermotolerance by a 25–38 °C upshift, then maintaining cells at 38 °C for 40 min, pma1.1 lowered subsequent tolerances of much higher lethal temperatures. Analysis of pulse-labelled S. cerevisiae proteins revealed reduced heat-shock protein synthesis in the pma1.1 mutant after a 25–38 °C heat shock. This may explain the greater increases in thermotolerance in wild-type as compared to pma1.1 cells after both were given identical 25–38 °C shocks. With more severe treatment (25–42 °C), heat-shock protein synthesis in wild-type cells, although initially high, was switched off more rapidly than in the pma1.1 mutant. These results indicate that plasma-membrane ATPase action exerts a major influence over several stress tolerances, as well as the extent and duration of heat-shock protein synthesis following induction of the heat-shock response.

Modulation of the Ca2+,Mg2+‐ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.

Vanadate-Resistant Mutants of Saccharomyces cerevisiae Show Alterations in Protein Phosphorylation and Growth Control

This work describes two spontaneous vanadate-resistant mutants of Saccharomyces cerevisiae with constitutive alterations in protein phosphorylation, growth control, and sporulation. Vanadate has been shown by a number of studies to be an efficient competitor of phosphate in biochemical reactions, especially those that involve phosphoproteins as intermediates or substrates. Resistance to toxic concentrations of vanadate can arise in S. cerevisiae by both recessive and dominant spontaneous mutations in a large number of loci. Mutations in two of the recessive loci, van1-18 and van2-93, resulted in alterations in the phosphorylation of a number of proteins. The mutant van1-18 gene also showed an increase in plasma membrane ATPase activity in vitro and a lowered basal phosphatase activity under alkaline conditions. Cells containing the van2-93 mutant allele had normal levels of plasma membrane ATPase activity, but this activity was not inhibited by vanadate. Both of these mutants failed to enter stationary phase, were heat shock sensitive, showed lowered long-term viability, and sporulated on rich medium in the presence of 2% glucose. The wild-type VAN1 gene was isolated and sequenced. The open reading frame predicts a protein of 522 amino acids, with no significant homology to any genes that have been identified. Diploid cells that contained two mutant alleles of this gene demonstrated defects in spore viability. These data suggest that the VAN1 gene product is involved in regulation of the phosphorylation of a number of proteins, some of which appear to be important in cell growth control.

Vanadate-resistant mutants of Saccharomyces cerevisiae show alterations in protein phosphorylation and growth control.

This work describes two spontaneous vanadate-resistant mutants of Saccharomyces cerevisiae with constitutive alterations in protein phosphorylation, growth control, and sporulation. Vanadate has been shown by a number of studies to be an efficient competitor of phosphate in biochemical reactions, especially those that involve phosphoproteins as intermediates or substrates. Resistance to toxic concentrations of vanadate can arise in S. cerevisiae by both recessive and dominant spontaneous mutations in a large number of loci. Mutations in two of the recessive loci, van1-18 and van2-93, resulted in alterations in the phosphorylation of a number of proteins. The mutant van1-18 gene also showed an increase in plasma membrane ATPase activity in vitro and a lowered basal phosphatase activity under alkaline conditions. Cells containing the van2-93 mutant allele had normal levels of plasma membrane ATPase activity, but this activity was not inhibited by vanadate. Both of these mutants failed to enter stationary phase, were heat shock sensitive, showed lowered long-term viability, and sporulated on rich medium in the presence of 2% glucose. The wild-type VAN1 gene was isolated and sequenced. The open reading frame predicts a protein of 522 amino acids, with no significant homology to any genes that have been identified. Diploid cells that contained two mutant alleles of this gene demonstrated defects in spore viability. These data suggest that the VAN1 gene product is involved in regulation of the phosphorylation of a number of proteins, some of which appear to be important in cell growth control.