Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Effect of nigericin on the H+-translocating adenosine triphosphatase from tonoplast of Hevea latex

Nigericin stimulated the ATPase activity of tightly-sealed membrane vesicles prepared from Hevea brasiliensis Müll.-Arg. lutoïds in the presence of K⁺. This stimulation required a functioning membrane since it was membrane-bound and since it was not observed for the ATPase activity solubilized from the tonoplast by dichloromethane. The extent of nigericin-induced stimulation of tonoplast ATPase was proportional to the ΔpH collapsed by the ionophore in the presence of K⁺.     

Kinetics of the purified plasma membrane H+-ATPase from red beet (Beta vulgaris)

The plasma membrane H⁺-ATPase (EC 3.6.1.35) was purified by washing red beet ( Beta vulgaris L.) plasma membranes with sodium deoxycholate and separating the ATPase, solubilized with lysophosphatidylcholine, by centrifugation in a glycerol gradient. The purified H⁺-ATPase had a sedimentation coefficient of about 8S. In the absence of exogenous protein substrates, the purified ATPase preparation did not present protein kinase activity. Compared with the H⁺-ATPase in the plasma membrane, the purified ATPase presented a higher affinity for adenosine 5'-triphosphate (ATP) and a lower sensitivity to the inhibitors vanadate and inorganic phosphate. These changes in the kinetics of the ATPase could also be observed by treating the membranes with lysophosphatidylcholine, without purifying the enzyme. These results can be explained assuming that lysophosphatidylcholine interacts with the ATPase altering its kinetics probably by stimulating the transformation from the inhibitor-binding conformation E2 into the ATP-binding conformation E1.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.     

Characterization of reconstituted plasma membrane H+-ATPase from maize roots

Corn ( Zea mays L.) plasma membranes from KI-washed microsomal fractions were further purified by isopycnic sucrose density centrifugation. An examination of separated fractions indicated that vesicles with nitrate-insensitive proton transport copurified with fractions containing vanadate-sensitive ATPase activity. The ATPase in purified plasma membrane was reconstituted into liposomes by a detergent dilution technique using deoxycholate. The reconstituted ATPase exhibited characteristics similar to those of the native enzyme. However, reconstituted preparations showed an enhanced sensitivity to vanadate, a diminished phosphatase activity and a high specific rate of ATP-dependent H⁺-transport. Apparent K_i values of reconstituted and native enzymes with respect to vanadate were 20 and 50 μ M , respectively; the KJ value of the H+-pumping of reconstituted ATPase was 30 μ M. The proton pumping of reconstituted vesicles could be discharged rapidly by p -trifluoromethoxyphenyl hydrazone (FCCP), hexokinase and vanadate. The hydrolysis of Mg-ATP by both native and reconstituted ATPases obeyed simple Michaelis-Menten plots with a K_m between 0.5 and 0.6 m M. The reconstituted ATPase retained a pH profile similar to that of native enzyme with a maximum of pH 6.5.     

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Ferredoxin-NADP⁺ reductase (FNR, EC I.18.1.2) from the green algae Chlorella fusca Shihira et Kraus 211–15, was purified to homogeneity. The molecular mass was 36.8 kDa as determined by SDS-polyacrylamide gel electrophoresis. The enzyme exhibits the typical spectrum of a flavoprotein with an absorption maximum at 459 nm and an A_273/459 ratio of 7.2. It contains one mol of FAD per mol of protein and the calculated extinction coefficient is 9.8 m M cm⁻¹. Four different forms of the purified enzyme were detected by isoelectric focusing (pI between 5.4 and 5.9), even when protease inhibitors were used during the first steps of the purification. Kinetic parameters were determined for several FNR-catalyzed reactions. NADP⁺ photoreduction gave comparable rates when either ferredoxin or flavodoxin was used.     

ATP-dependent H+-pumping of a low-density fraction of pea stem microsomes

A low-density fraction of pea ( Pisum sativum L. cv. Alaska) stem microsomes, obtained from a discontinuous sucrose gradient, possessed an H⁺-ATPase able to generate a proton gradient and an electrical potential. The proton pumping was insensitive to monovalent cations, to vanadate and oligomycin, required a permeant anion and was inhibited by nitrate, N, N'-dicyclohexylcarbodiimide and diethylstilbestrol. The H⁺-ATPase had a pH optimum around 6.0–6.5 and was saturable with respect to the substrate Tris-ATP (K_m≅ 0.4 m M ). Ca²⁺ (0.05–1 m M ) induced a dissipation of the ATP-generated δpH without affecting ATPase activity. At physiological concentrations (1–5 m M ), nitrate caused an initial slight increase of the ATP-generated proton gradient followed by a complete dissipation after 2–3 min. The dissipating effect was not caused by inhibition of ATPase activity, since ATP prevented the nitrate-induced collapse of δpH. On the other hand, ATPase activity, evaluated as release of P_i, was not inhibited by concentrations lower than 20 m M KNO₃. These results indicate that nitrate entered the vesicles in response to an electrical potential and then could exit in symport with protons, while Ca²⁺ entered in exchange for protons (antiport).     

Temperature dependence of the barley root plasma membrane-bound Ca2+- and Mg2+-dependent ATPase

Arrhenius plots of the maximal velocities for the Ca²⁺-and Mg²⁺-dependent ATPase activities found in a plasma membrane-rich microsome fraction isolated from the roots of barley ( Hordeum vulgare L. cv. Conquest) were nonlinear. Arrhenius plot analyses using a relation which produced curvilinear Arrhenius plots accurately fit the data and allowed the calculation of the activation enthalpies and molar heat capacities of activation. The temperature dependence of the computed Km values for the Ca²⁺- and Mg²⁺-dependent ATPase activities was complex, with the highest enzyme-substrate affinities being obtained near the barley seedling growth temperature (16°C). Using electron paramagnetic resonance spectroscopy with amphiphilic cationic and anionic spin probes, it was possible to demonstrate that temperature changes and increasing Ca²⁺ concentrations could alter the mobility of the membrane lipid polar head groups. Inhibition of the ATPase activities by high levels of Ca²⁺ may result from a Ca2+-induced reduction in the lipid polar head group mobility. The possible role of lipid polar head group-protein interactions in the complex temperature dependence of the barley root ATPase kinetic constants is discussed.     

Effects of Ca2+ and polyamines on Na+ and Rb+ influx in excised maize roots

When 1 m M spermidine or spermine was included in an absorption solution which contained 20 m M Na⁺ and 1 m M Rb⁺, Na⁺ influx into excised maize roots ( Zea mays L. cv. Golden Cross Bantam) was reduced. Rb⁺ influx was reduced in the presence of spermidine and uneffected in the presence of spermine when compared with control solutions. When 1 m M Ca²⁺ replaced the polyamines, Na⁺ influx was strongly reduced and Rb⁺ influx was promoted. Rb⁺ influx from 1 m M Rb⁺ solutions which did not contain Na⁺ was also promoted by 1 m M Ca²⁺, but was inhibited by 1 m M spermidine. This Ca²⁺ promotion of Rb⁺ influx could be reversed by 10 times greater concentration of spermidine in the absorption solution. H⁺ efflux from excised roots was inhibited by spermidine when compared with Ca²⁺ or control solutions, however, the plasma membrane ATPase was not inhibited by spermidine. It is concluded that external Ca²⁺ plays two separate roles in membrane function, only one of which can be substituted for by polyamines. The first role, maintenance of membrane integrity, can be substituted for by spermidine or spermine. The second function, maintenance of the Rb⁺ transport mechanism, is Ca²⁺ specific and cannot be substituted for by spermidine or spermine. The results of this study are discussed in terms of electrostatic interactions between the plasma membrane and the Ca²⁺ or polyamines.     

Bacterial phytotoxins, syringomycin, syringostatin and syringotoxin, exert their effect on the plasma membrane H+-ATPase partly by a detergent-like action and partly by inhibition of the enzyme

Syringostatin is a newly discovered phytotoxin produced by a phytopathogenic bacterium Pseudomonas syringae pv. syringae lilac isolate. The effects of syringostatin and the similar phytotoxins, syringomycin and syringotoxin, on H⁻-ATPase activity were investigated using cultured mung bean ( Vigna radiata L. cv. Ryokuto) cells or plasma membrane vesicles isolated from mung bean hypocotyls. ³¹P-NMR analysis of cultured cells treated with syringostatin revealed that the cytoplasmic pH was decreased. When plasma membrane was prepared by a two-step method (Dextran gradient followed by a sucrose gradient). syringostatin, syringomycin and syringotoxin inhibited the H⁺-ATPase activity in a dose-dependent manner. In contrast, these toxins stimulated H⁺-ATPase activity when plasma membrane was prepared by a one-step method (sucrose gradient). While these toxins inhibited the H⁺-ATPase activity of inside-out plasma membrane vesicles, the H⁺-ATPase activity of right-side-out vesicles was stimulated. The detergent. Triton X-100, abolished this stimulatory effect of the toxins on the H⁺-ATPase of right-side-out vesicles and of one-step purified plasma membrane. The toxins also inhibited the activity of the plasma membrane H⁺-ATPase solubilized with deoxycholate and Zwittergent 3–14. Taken together, these results indicate that these toxins exert their effects partly by a detergent-like action on the plasma membrane and partly by inhibition of the enzyme.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots

Physiological and biochemical modifications induced by Fe-deficiency have been studied in cucumber ( Cucumis sativus L. cv. Marketer) roots, a Strategy I plant that initiates a rapid acidification of the medium and an increase in the electric potential difference when grown under Fe-deficiency. Using the aqueous two-phase partitioning method, a membrane fraction which has the plasmalemma characteristics was purified from roots of plants grown in the absence and in the presence of iron. The plasma membrane vesicles prepared from Fe-deficient plants showed an H⁺-ATPase activity (EC 3.6.1.35) that is twice that of the non-deficient control. Furthermore, membranes from Fe-deficient plants showed a higher capacity to reduce Fe³⁺-chelates. The difference observed in the reductase activity was small with ferricyanide (only 30%) but was much greater with Fe³-EDTA and Fe³-citrate (210 and 250%, respectively). NADH was the preferred electron donor for the reduction of Fe³⁺ compounds. Fe³⁺ reduction in plasma membrane from cucumber roots seems to occur with utilisation of superoxide anion, since addition of superoxide dismutase (SOD; EC 1.15.1.1) "in vitro" decreased Fe³⁺ reduction by 60%.
The response and the difference induced by iron starvation on these two plasma membrane activities together with a possible involvement of O₂ in controlling the Fe³⁺/Fe²⁺ ratio in the rhizosphere are discussed.     

Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet

ABSTRACT. We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca²⁺ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca²⁺ uptake by digitonin-permeabilired epimastigotes. Crystal violet also induced Ca²⁺ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca²⁺ uptake and the (Ca²⁺-Mg²⁺)ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca²⁺ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

V- AND P-TYPE Ca2+-STIMULATED ATPases IN A CALCIFYING STRAIN OF PLEUROCHRYSIS SP. (HAPTOPHYCEAE)

Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane-bound Ca²⁺-stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca²⁺-stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca²⁺-stimulated ATPase. The Ca²⁺-stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP-dependent H⁺ movement, but not ⁴⁵Ca²⁺ transport, across the coccolith vesicle was demonstrated. The Ca²⁺-stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP-dependent, vanadate-sensitive efflux of ⁴⁵Ca²⁺ was demonstrated for microsomal material derived from gradient-isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross-reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross-react. These findings show that a V-type Ca²⁺-stimulated ATPase is located on the coccolith vesicle membrane and a P-type Ca²⁺-stimulated ATPase is located on the plasma membrane.