Mitochondrial H+-ATPase: Identification of Subunits of the F0 Subcomplex that Contact Membrane Lipids

The subunits of the F₀ membrane sector of bovine heart mitochondrial H⁺-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H⁺-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-¹⁴C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([¹²⁵I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F₀ membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry.     

Butyricin 7423 and the membrane H+-ATPase of Clostridium pasteurianum

The bacteriocin butyricin 7423 inhibited the activity of the membrane H⁺-ATPase (BF₀, F₁) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF₁ component. In vitro studies with the H⁺-ATPases of mutant strains selected for diminished sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BF₀ component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H⁺-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular ⁸⁶Rb⁺ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H⁺-ATPase. High extracellular concentrations of K⁺ ions enabled Cl. pasteurianum to survive exposure to low concentrations of this membrane-active bacteriocin.Non-standard abbreviations H⁺-ATPase proton translocating adenosine 5-triphosphatase (EC 3.6.1.3) - DCCD dicyclohexylcarbodiimide     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Subunit composition,biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase

The yeast vacuole is acidified by a vacuolar proton-translocating ATPase (H⁺-ATPase) that closely resembles the vacuolar H⁺-ATPases of other fungi, animals, and plants. The yeast enzyme is purified as a complex of eight subunits, which include both integral and peripheral membrane proteins. The genes for seven of these subunits have been cloned, and mutant strains lacking each of the subunits (vma mutants) have been constructed. Disruption of any of the subunit genes appears to abolish the function of the vacuolar H⁺-ATPase, supporting the subunit composition derived from biochemical studies. Genetic studies of vacuolar acidification have also revealed an additional set of gene products that are required for vacuolar H⁺-ATPase activity, but may not be part of the final enzyme complex. The biosynthesis, assembly, and targeting of the enzyme is being elucidated by biochemical and cell biological studies of thevma mutants. Initial results suggest that the peripheral and integral membrane subunits may be independently assembled.     

Expression of the Catalytic Subunit of Ouabain-Sensitive H+,K+-ATPase in Rat Skin Epidermis

A comparative localization of Na⁺,K⁺-ATPase and ouabain-sensitive H⁺,K⁺-ATPase in rat skin was performed using in situ RNA hybridization and immunohistochemistry. Na⁺,K⁺-ATPase was predominantly detected in the basal layer of the epithelium, whereas the ouabain-sensitive H⁺,K⁺-ATPase, in the granular and prickle cell layers. The genes of these ATPases are thus expressed in epithelial cells at different stages of their development. The hypothesis was advanced that the ouabain-sensitive H⁺,K⁺-ATPase is involved in maintaining the skin pH value. The probes specific to the mRNAs of the full-size -subunit of the ouabain-sensitive H⁺,K⁺-ATPase and its truncated form were used to establish a similar distribution of both mRNA variants in skin.     

Structure and function of the yeast vacuolar membrane proton ATPase

Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H⁺-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F₀F₁-type ATP synthase and the plasma membrane E₁E₂-type H⁺-ATPase. The vacuolar membrane H⁺-ATPase is composed of three major subunits, subunita (M _r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H⁺-ATPase under two kinetic conditions have been determined to be 0.9–1.1×10⁵ daltons for single-cycle hydrolysis of ATP and 4.1–5.3×10⁵ daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H⁺-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP carbonyl cyanidem-chlorophenyl hydrazone - DCCD N,N-dicyclohexylcarbondiimide - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Pi inorganic phosphate - SDS sodium dodecylsulfate - SF6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile - SITS 4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid - ZW3-14 N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate     

Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells. Plasma membrane-type (vanadate-sensitive) H⁺-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes. A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H⁺-ATPase (designated BHA1) has been cloned. BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H⁺-ATPase. BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants. It belongs to the subfamily of plasma membrane H⁺-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes. In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells. These results were confirmed by immunocytochemistry. The relatively low expression of plasma membrane-type H⁺-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes. Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.     

Effects of colchicine on the accumulation of vacuolar H+-pyrophosphatase and H+-ATPase in germinating Acacia mangium seeds and the recovery effects by sucrose, indole butyric acid and 6-benzyladenine

Plant vacuoles were isolated from cotyledons of germinatingAcacia mangium seeds, which had been treated with or withoutcolchicine, to measure vacuolar membrane pyrophosphate (PPi)- andATP-dependent H⁺ transport activities, and enzymaticactivities of H⁺-pyrophosphatase(H⁺-PPase) and H⁺-ATPase. Innon-colchicine-treated seeds, activities of the two enzymes increasedrapidly after seed germination to almost a maximal level on the seventhday. A linear function relationship exists in magnitude between PPi- orATP-dependent H⁺transport activity and its correspondingenzymatic activity. The former regression equation is: PPi-dependentH⁺ transport activity(%A.min^–1.g^–1) =–0.039 + H⁺-PPase activity(units.mg^–1) × 1.574, the latter is:ATP-dependent H⁺ transport activity(%A.min^–1.g^–1) =–0.003 + H⁺-ATPase activity(units.mg^–1) × 0.549. In colchicine-treatedseeds, activities of the two enzymes increased very slowly during 8 daysof germination and the relationship to their respectiveH⁺ transport activities was not in agreement with theabove-mentioned regression equations. PPi- and ATP-dependentH⁺ transport activities were lower than thecorresponding values calculated from H⁺-PPase activityand H⁺-ATPase activity according to the two regressionequations, respectively. However, when sucrose, indole butyric acid(IBA), or 6-benzyladenine (6-BA) were applied exogenously to the seedsfollowing colchicine treatment for 3 days, activities ofH⁺-PPase, H⁺-ATPase, PPi- andATP-dependent H⁺ transport in the 6-day-old seedlingsall increased. By statistical analysis, it was concluded that colchicineinhibits cotyledon vacuolar membrane H⁺-PPase,H⁺-ATPase activities, PPi- and ATP-dependentH⁺ transport activities during seed germination andearly seedling growth of Acacia mangium. The inhibitory effectsof colchicine could be overcome by IBA, 6-BA and sucrose to varyingdegrees.     

Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

Dependence of the kinetics of secondary active transports in yeast on H+-ATPase acidification

Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H⁺-ATPase, was inhibited to different degrees by D₂O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide. The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions. An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid. In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mm KC1 had no effect on the transport rates. The ATPase-deficient mutant S. cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and pH were unchanged.Other types of acidification (spontaneous upon suspension; K⁺ stimulated) did not affect the secondary uptake systems.     

Dimorphism-associated changes in plasma membrane H+-ATPase activity of Candida albicans

In situ plasma membrane H⁺-ATPase activity was monitored during pH-regulated dimorphism of Candida albicans using permeabilized cells. ATPase activity was found to increase in both the bud and germ tube forming populations at 135 min which coincides with the time of evagination. Upon reaching the terminal phenotype the mycelial form exhibited higher H⁺-ATPase activity as compared to the yeast form. At the time of evagination H⁺-efflux exhibited an increase. K⁺ depletion resulted in attenuated ATPase activity and glucose induced H⁺-efflux. The results demonstrate that ATPase may play a regulatory role in dimorphism of C. albicans and K⁺ acts as a modulator.Abbreviations PM Plasma membrane - pHi intracellular pH - Pi inorganic phosphorus - TET Toluene: Ethanol: Triton X-100     

Probing the structure of the Neurospora crassa plasma membrane H+-ATPase

The structure of the Neurospora crassa plasma membrane H⁺-ATPase has been investigated using a variety of chemical and physicochemical techniques. The transmembrane topography of the H⁺-ATPase has been elucidated by a direct, protein chemical approach. Reconstituted proteoliposomes containing purified H⁺-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin, and the numerous peptides released were purified by HPLC and subjected to amino acid sequence analysis. In this way, seventeen released peptides were unequivocally identified as located on the cytoplasmic side of the membrane, and numerous intervening segments could be inferred to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return between sequences established to be cytoplasmically located. Additionally, three large membrane-embedded segments of the H⁺-ATPase were isolated using our recently developed methods for purifying hydrophobic peptides, and identified by amino acid sequence analysis. This information established the topographical location of virtually all of the 919 residues in the H⁺-ATPase molecule, allowing the formulation of a reasonably detailed model for the transmembrane topography of the H⁺-ATPase polypeptide chain. Separate studies of the cysteine chemistry of the H⁺-ATPase have demonstrated the existence of a single disulfide bridge in the molecule, linking the NH₂- and COON-terminal membrane-embedded domains. And, analyses of the circular dichroism and infrared spectra of the purified H⁺-ATPase have elucidated the secondary structure composition of the molecule. A first-generation model for the tertiary structure of the H⁺-ATPase based on this information and other considerations is presented.     

Fluorescent labelling of Na+,K+-ATPase in intact cells by use of a fluorescent derivative of ouabain: Salinity and teleost chloride cells

Summary Anthroylouabain, a fluorescent derivative of ouabain, was used to localize Na⁺,K⁺-ATPase in transport epithelia of two species of teleosts. Exposure of the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) and the jaw skin of the long-jawed mudsucker (Gillichthys mirabilis) to a 2 M anthroylouabain solution resulted in the appearance of cells stained bright blue. These were deemed to be chloride cells by their large size, distinct morphology and co-localization of DASPEI fluorescence, a mitochondrial stain. Addition of ouabain (1 mM final concentration) greatly decreased anthroylouabain fluorescent staining of chloride cells of seawater-adapted fish. Exposure of opercular membranes from freshwater tilapia to 2 M anthroylouabain did not result in significant staining. Anthroylouabain is therefore a useful vital stain for localizing Na⁺,K⁺-ATPase in chloride cells of seawater-adapted teleosts, and may be useful for fluorescent labelling of ouabain-sensitive Na⁺,K⁺-ATPase in other tissues and species.     

Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini

With a homologous gene region we successfully isolated a Na⁺/H⁺ antiporter gene from a halophytic plant, Atriplex gmelini, and named it AgNHX1. The isolated cDNA is 2607 bp in length and contains one open reading frame, which comprises 555 amino acid residues with a predicted molecular mass of 61.9 kDa. The amino acid sequence of the AgNHX1 gene showed more than 75% identity with those of the previously isolated NHX1 genes from glycophytes, Arabidopsis thaliana and Oryza sativa. The migration pattern of AgNHX1 was shown to correlate with H⁺-pyrophosphatase and not with P-type H⁺-ATPase, suggesting the localization of AgNHX1 in a vacuolar membrane. Induction of the AgNHX1 gene was observed by salt stress at both mRNA and protein levels. The expression of the AgNHX1 gene in the yeast mutant, which lacks the vacuolar-type Na⁺/H⁺ antiporter gene (NHX1) and has poor viability under the high-salt conditions, showed partial complementation of the NHX1 functions. These results suggest the important role of the AgNHX1 products for salt tolerance.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

Expression of theunc genes inEscherichia coli

Theunc (or atp) operon ofEscherichia coli comprises eight genes encoding the known subunits of the proton-translocating ATP synthase (H⁺-ATPase) plus a ninth gene (uncI) of unknown function. The subunit stoichiometry of the H⁺-ATPase ( ₃₃₁₁₁a₁b₂c_10–15) requires that the respectiveunc genes be expressed at different rates. This review discusses the experimental methods applied to determining how differential synthesis is achieved, and evaluates the results obtained. It has been found that the primary level of control is translational initiation. The translational efficiencies of theunc genes are determined by primary and secondary mRNA structures within their respective translational initiation regions. The respective rates of translation are matched to the subunit requirements of H⁺-ATPase assembly. Finally, points of uncertainty remain and experimental strategies which will be important in future work are discussed.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Analysis of the H+/sugar symport in yeast under conditions of depolarized plasma membrane

H⁺/sugar symport in the obligatory aerobic yeastRhodotorula glutinis was analyzed under conditions where the plasma membrane was selectively depolarized by the lipophilic cation tetraphenylphosphonium (TPP⁺). Control experiments showed that this treatment did not impair the transmembrane pH, the cell energy charge, and the function of plasma membrane H⁺-ATPase. The kinetic data were fitted to elementary functions derived from a model constructed on the basis of some simplifying premises for ordered (either C + H⁺ + S or C + S + H⁺) and random reaction mechanisms. In addition, the comparison of the kinetic parameters in fully energized and depolarized cells provided information about the free carrier charge. It was concluded that the binding sequence of formation of the ternary carrier/H⁺/substrate complex follows a random mechanism and that the carrier bears a negative charge.     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

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 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 -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.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol