pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H(+) ions and thereby regulates its cytosolic pH (pH(i)), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH(i) and to recover from an imposed intracellular acidification in the absence of extracellular Na(+), thus ruling out the involvement of a Na(+)/H(+) exchanger in both processes. Both phenomena were ATP-dependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH(i) of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na(+)/H(+) exchange, had relatively little effect. The resting pH(i) of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N'-dicyclohexylcarbodiimide, and by bafilomycin A(1), a potent inhibitor of V-type H(+)-ATPases. Bafilomycin A(1) blocked pH(i) recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H(+)-ATPase, which serves as the major route for the efflux of H(+) ions.     

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.     

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.     

Involvement of plasma membrane H+-ATPase in anoxic elongation of stems in pondweed (Potamogeton distinctus) turions

? Pondweed (Potamogeton distinctus) turions can elongate in the absence of O(2). Alcoholic fermentation serves to produce energy for anoxic elongation via the breakdown of starch stored in cells. However, the mechanism of cell growth during anoxic elongation is not fully understood. ? Changes in pH, H(+) equivalent and lactate content of the incubation medium were measured during anoxic elongation. The effects of fusicoccin (FC), indole-3-acetic acid (IAA), vanadate, erythrosine B and K(+) channel blockers on anoxic elongation were examined. Cytoplasmic pH and vacuolar pH were measured by (31)P nuclear magnetic resonance (NMR) spectroscopy. ? Acidification of the incubation medium occurred during anoxic elongation. The contribution of CO(2) and lactic acid was not sufficient to explain the acidification. FC and IAA enhanced the elongation of stem segments. Vanadate and erythrosine B inhibited anoxic elongation. Acid growth of notched segments was observed. The activity of plasma membrane H(+)-ATPase extracted from pondweed turions was increased slightly in anoxic conditions, but that from pea epicotyls sensitive to anoxic conditions was decreased by incubation in anoxic conditions. Both the cytoplasmic pH and vacuolar pH of pondweed turion cells chased by (32)P NMR spectroscopy were stabilized during a short period < 3 h after anoxic conditions. ? We propose that the enhancement of H(+) extrusion by anoxic conditions induces acidification in the apoplast and may contribute to the stabilization of pH in the cytoplasm.     

The yeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alter pH homeostasis

The BTN1 gene product of the yeast Saccharomyces cerevisiae is 39% identical and 59% similar to human CLN3, which is associated with the neurodegenerative disorder Batten disease. Furthermore, btn1-Delta strains have an elevated activity of the plasma membrane H(+)-ATPase due to an abnormally high vacuolar acidity during the early phase of growth. Previously, DNA microarray analysis revealed that btn1-Delta strains compensate for the altered plasma membrane H(+)-ATPase activity and vacuolar pH by elevating the expression of the two genes HSP30 and BTN2. We now show that deletion of either HSP30 or BTN2 in either BTN1(+) or btn1-Delta strains does not alter vacuolar pH but does lead to an increased activity of the vacuolar H(+)-ATPase. Deletion of BTN1, BTN2, or HSP30 does not alter cytosolic pH but diminishes pH buffering capacity and causes poor growth at low pH in a medium containing sorbic acid, a condition known to result in disturbed intracellular pH homeostasis. Btn2p was localized to the cytosol, suggesting a role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase. Increased expression of HSP30 and BTN2 in btn1-Delta strains and diminished growth of btn1-Delta, hsp30-Delta, and btn2-Delta strains at low pH reinforce our view that altered pH homeostasis is the underlying cause of Batten disease.     

Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, stimulates the transport activity

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mm ZnCl(2). Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope (65)Zn(2+). Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H(+)-ATPase. The activity was inhibited by bafilomycin A(1), an inhibitor of the H(+)-ATPase. The K(m) for Zn(2+) and V(max) of AtMTP1 were determined to be 0.30 microm and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn(2+) and resistant to Co(2+). The K(m) and V(max) values were increased 2-11-fold. These results indicate that AtMTP1 functions as a Zn(2+)/H(+) antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn(2+) and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn(2+).     

The intracellular dissipation of cytosolic calcium following glucose re-addition to carbohydrate depleted Saccharomyces cerevisiae

Glucose re-addition to carbohydrate starved yeast cells leads to a transient elevation of eytosolic calcium (TECC). Concomitantly, a cytosolic proton extrusion occurs through the activation of the vacuolar H(+)-ATPase and the plasma membrane H(+)-ATPases. This study addressed the dissipation of the TECC through intracellular compartmentalization and the possible affects of the H(+)-ATPases on this process. Both the vacuole and the Golgi-ER apparatus were found to play important roles in distributing calcium to internal stores. Additionally, the inhibition of cytosolic proton extrusion augmented cytosolic calcium responses. A model where pH dependent cytosolic calcium buffering plays an important role in the dissipation of the TECC in Saccharomyces cerevisiae is proposed.     

Sodium transport in plant cells

Salinity limits plant growth and impairs agricultural productivity. There is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole-plant levels, however, the mechanisms of sodium transport appear to be fundamentally similar. At the cellular level, sodium ions gain entry via several plasma membrane channels. As cytoplasmic sodium is toxic above threshold levels, it is extruded by plasma membrane Na(+)/H(+) antiports that are energized by the proton gradient generated by the plasma membrane ATPase. Cytoplasmic Na(+) may also be compartmentalized by vacuolar Na(+)/H(+) antiports. These transporters are energized by the proton gradient generated by the vacuolar H(+)-ATPase and H(+)-PPiase. Here, the mechanisms of sodium entry, extrusion, and compartmentation are reviewed, with a discussion of recent progress on the cloning and characterization, directly in planta and in yeast, of some of the proteins involved in sodium transport.     

Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages

The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.     

The vacuolar H+ -ATPase mediates intracellular acidification required for neurodegeneration in C. elegans

Numerous studies implicate necrotic cell death in devastating human pathologies such as stroke and neurodegenerative diseases. Investigations in both nematodes and mammals converge to implicate specific calpain and aspartyl proteases in the execution of necrotic cell death. It is believed that these proteases become activated under conditions that inflict necrotic cell death. However, the factors that modulate necrosis and govern the erroneous activation of these otherwise benign enzymes are largely unknown. Here we show that the function of the vacuolar H(+)-ATPase, a pump that acidifies lysosomes and other intracellular organelles, is essential for necrotic cell death in C. elegans. Cytoplasmic pH drops in dying cells. Intracellular acidification requires the vacuolar H(+)-ATPase, whereas alkalization of endosomal and lysosomal compartments by weak bases protects against necrosis. In addition, we show that vacuolar H(+)-ATPase activity is required downstream of cytoplasmic calcium overload during necrosis. Thus, intracellular pH is an important modulator of necrosis in C. elegans. We propose that vacuolar H(+)-ATPase activity is required to establish necrosis-promoting, acidic intracellular conditions that augment the function of executioner aspartyl proteases in dying cells. Similar mechanisms may contribute to necrotic cell death that follows extreme acidosis-for example, during stroke-in humans.     

The dual mechanism of the antifungal effect of new lysosomotropic agents on the Saccharomyces cerevisiae RXII strain

Quinacrine was used to visualize the intracellular pH changes in the yeast strain Saccharomyces cerevisiae RXII occurring after exposure to four recently-synthesized lysosomotropic drugs: DM-11, PY-11, PYG-12s and DMAL-12s. The cells took up quinacrine, mostly accumulating it in their vacuoles. DM-11 and PY-11 gave rise to diffuse quinacrine fluorescence throughout the cells, with the vacuoles staining to a somewhat greater extent than the cytosol. This quinacrine-detected overall acidification of the cell interior is very probably caused by blocking of plasma membrane H(+)-ATPase. PYG-12s gave rise to a strong vacuolar accumulation of the dye. Like the vacuolar ATPase inhibitor bafilomycin A(1), DMAL-12s strongly lowered the intensity of quinacrine fluorescence. Owing to its low pK(a), it can penetrate rapidly into the cells and may inhibit vacuolar H(+)-ATPase and prevent quinacrine-detectable vacuolar acidification without causing strong cell acidification. Since these drugs were found to penetrate into the cells, their lack of effect may reflect a higher resistance of both plasma membrane H(+)-ATPase and vacuolar ATPase to the drugs. Our data indicate that the lysosomotropic drugs under study have a dual action. On entering the cell, they cause intracellular acidification, very probably by inhibiting plasma membrane H(+)-ATPase and curtailing active proton pumping from the cells. Furthermore, they interfere with the function of V-type ATPase, causing vacuolar alkalinization and eventually cell death.     

Ion metabolism in malaria-infected erythrocytes

Malaria parasites of the genus Plasmodium spend much of their asexual life cycle inside the erythrocytes of their vertebrate hosts. Parasites presumably have to exploit metabolic and transport mechanisms to adapt themselves to the host erythrocyte's physicochemical environment. This review surveys the metabolism and transport of Ca2+, alkali cations, and H+ in malaria-infected erythrocytes. The Ca2+ content of Plasmodium-infected erythrocytes increases as the parasite matures. An increase in the influx of extracellular Ca2+ into infected erythrocytes is evident at later stages of parasite development. In infected erythrocytes, Ca2+ is almost exclusively localized in the parasite compartment and changes but little in the cytosol of the host cell. The importance of Ca2+ in supporting the growth of intraerythrocytic parasites and the invasion of erythrocytes by the merozoite has been assessed by depletion of extracellular Ca2+ with chelators, or by disturbance of the metabolism and transport of Ca2+ with a variety of Ca2+ modulators. Membranes of malaria-infected erythrocytes change their permeability to alkali cations. Hence, levels of K+ decrease and levels of Na+ increase in the cytosol of infected erythrocytes. Intraerythrocytic parasites maintain a high K+, low Na+ state, suggesting a mechanism for transporting K+ inward and Na+ outward against concentration gradients of the alkali cations across the parasite plasma membrane and/or the parasitophorous vacuole membrane (PVM). Concomitantly, P. falciparum can grow in Na(+)-enriched human erythrocytes. Experimental evidence suggests that Plasmodium possesses in its plasma membrane a proton pump which is very sensitive to orthovanadate, carbonylcyanide m-chlorophenylhydrazone, a protonophore, and dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, but is only slightly sensitive to inhibitors of bacterial and mitochondrial respiration, such as antimycin A, CN-, or N3-, and ouabain, a Na+, K(+)-ATPase inhibitor. By operating this proton pump, parasites extrude H+ and thus generate an electrochemical gradient of protons (an internal negative membrane potential and a concentration gradient of protons) across the parasite plasma membrane. The electrochemical gradient apparently drives inward movement of Ca2+ and, possibly, glucose from the cytosol of infected erythrocytes. Little is known about the transport properties of the PVM. Recent sequence studies suggest that Plasmodium contains a cation-transporting ATPase which exhibits a high homology to the Ca2(+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae by addition of hydrogen peroxide.

Addition of hydrogen peroxide (greater than 10 mM) to aerated derepressed cells of S. cerevisiae in the absence of substrate caused a boost of endogenous respiration and both intra- and extracellular acidification, without any significant change in cellular ATP level. Furthermore, a hyperpolarization of the plasma membrane was indicated by an enhanced accumulation of tetraphenylphosphonium in the cells. The extracellular pH attained was as low as 3.5. The acidification could be suspended by the H(+)-ATPase inhibitors diethylstilbestrol and dicyclohexylcarbodiimide and was, in general, associated with an opposite flux of K+. K+ also stimulated the H(+)-ATPase activity in the purified plasma membrane fraction. These results are consistent with the plasma membrane H(+)-ATPase being involved in the H+ extrusion induced by H2O2 in the absence of substrate. Extended exposure of cells to H2O2 led eventually to an arrest of both respiration and ion fluxes that could be again lifted by depolarizing the plasma membrane. Along with differences in the cellular NADH/NAD+ ratio and in the participation of organic acids, this makes the H2O2-induced acidification distinct from that induced by glucose.     

A cytosolic inhibitor of vacuolar H(+)-ATPases from mammalian kidney.

Regulation of the vacuolar H(+)-ATPase in organellar and transepithelial acidification has been attributed to the effects of the proton electrochemical gradient across the membrane or to changes in the number of proton pumps. We now report the identification and purification of a protein from bovine kidney cytosol that inhibits both ATPase activity and proton translocating activity of vacuolar H(+)-ATPases. Its relative molecular weight (M(r)) is 6300, similar to that for protein inhibitors of the mitochondrial F0F1-ATPase. The newly identified cytosolic inhibitor protein may participate in the physiologic regulation of the vacuolar H(+)-ATPase by suppressing activity directly.     

A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.     

Acylspermidine derivatives isolated from a soft coral,Sinularia sp,inhibit plant vacuolar H(+)-pyrophosphatase

H(+)-pyrophosphatase (H(+)-PPase), which pumps H(+) across membranes coupled with PP(i) hydrolysis, is found in most plants, and some parasitic protists, eubacteria and archaebacteria. We assayed a number of extracts derived from 145 marine invertebrates as to their inhibitory effect on plant vacuolar H(+)-PPase. Acylspermidine derivatives [RCONH(CH(2))(3)N(CH(3))(CH(2))(4)N(CH(3))(2)] from a soft coral (Sinularia sp.) inhibited the PPi-hydrolysis activity of purified H(+)-PPase and the PP(i)-dependent H(+) pump activity (half inhibition concentration, 1 micro M) of vacuolar membranes of mung bean. The apparent K(i) was determined to be 0.9 micro M. Acylspermidines did not affect the activity of vacuolar H(+)-ATPase, plasma membrane H(+)-ATPase, mitochondrial ATPase or cytosolic PPase. Acylspermidines inhibited the acidification of vacuoles in protoplasts, as found on monitoring by the acridine orange fluorescent method. These results indicate that acylspermidine derivatives represent new inhibitors of H(+)-PPase with relatively high specificity.     

The HPV-16 E5 oncogene and bafilomycin A(1) influence cell motility

It is known that the proper function of the vacuolar H(+)-ATPase is inhibited by bafilomycin A(1). In transfected cells the E5 protein interacts with the 16 kDa subunit of the vacuolar H(+)-ATPase. Thereby the pH gradient in endocytic structures is impaired. The present study demonstrates for the first time that the inhibition of the vacuolar H(+)-ATPase in NIH3T3 cells with bafilomycin A(1) or by transfection of cells with the HPV-16 E5 oncogene leads to a changed morphology and a reduced motility as shown by computer-assisted video recordings and image analysis. Bafilomycin A(1) potentiates the effect of the E5 protein on cell motility and this cooperative effect indicates that the E5 protein and bafilomycin A(1) either target the vacuolar H(+)-ATPase differently or that the E5 protein has additional targets in transfected cells. Our data therefore show that proper function of the vacuolar H(+)-ATPase is needed for normal cell locomotion.     

Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies

We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H(+)-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g(-1) cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H(+) ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H(+) extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H(+)-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H(+)-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.