Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma membrane vesicles

Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H⁺-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H⁺ was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H⁺-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, K_m for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H⁺-ATPase activity in vivo.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

Dissociation and Reassembly of the Vacuolar H-ATPase Complex from Oat Roots

Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H⁺-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H⁺-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg²⁺ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H⁺-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO₃, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO₃ > KBr > K-acetate > K₂SO₄ > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H⁺-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H⁺ pumping as seen from the recovery of H⁺ transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H⁺-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H⁺-ATPase to form a functional H⁺ pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H⁺-ATPase in vivo.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase

The H⁺/ATP stoichiometry was determined for an anion-sensitive H⁺-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A₃₄₀) or by monitoring the release of ³²P from [γ-³²P]ATP. Inasmuch as this anion-sensitive H⁺-ATPase is strongly inhibited by NO₃⁻, initial rates of H⁺ influx and ATP hydrolysis were measured in the absence and presence of NO₃⁻ to account for ATPase activity not involved in H⁺ transport. The NO₃⁻-sensitive activities were calculated and used to estimate the ratio of H⁺ transported to ATP hydrolyzed. These measurements resulted in an estimate of the H⁺/ATP stoichiometry of 1.96 ± 0.14 suggesting that the actual stoichiometry is 2 H⁺ transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H⁺ across the tonoplast measured in vivo, our result suggests that the H⁺-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.     

Adaptation of plasma membrane H+ ATPase and H+ pump to P deficiency in rice roots

The plasma membrane (PM) H⁺ ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice (Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H⁺ ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H⁺ ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value (K _m ) of PM H⁺ ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H⁺ ATPase in P deficient roots was related to a slightly increase of PM H⁺ ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H⁺-ATPase and H⁺ pump, which contributed to the rhizosphere acidification in rice roots.     

Differential Inhibition of Tonoplast H-ATPase Activities by Fluorescamine and Its Derivatives

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H⁺-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H⁺ leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.     

High-Pressure Effects on Vacuolar H+-ATPase from Etiolated Mung Bean Seedlings

A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H⁺-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H⁺-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both V _max and K _m of solubilized vacuolar H⁺-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg²⁺-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H⁺-ATPase by pressure may result from changes in protein–protein interaction. The conformational change of solubilized vacuolar H⁺-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H⁺-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit–subunit interaction is crucial for the integrity and the function of plant vacuolar H⁺-ATPase. It is also suggested that the assembly of the vacuolar H⁺-ATPase complex is probably not random, but follows a sequestered pathway.     

Active vanadate-sensitive H+ translocation in corn roots membrane vesicles and proteoliposomes

A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min⁻¹ · mg⁻¹, depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent K_m-value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H⁺-pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I₅₀ = 2 μM) or to SW 26 (I₅₀ = 0.5 μM) than that of the original membrane fractions.     

Factors associated with the instability of nitrate-insensitive proton transport by maize root microsomes

Proton transport catalyzed by the nitrate-insensitive, vanadate-sensitive H⁺-ATPase in microsomes from maize (Zea mays L.) roots washed with 0.25 molar KI decreased as a function of time at 0 to 4°C. The rate of proton transport was approximately one-half of that by freshly isolated microsomes after 6 to 18 hours of cold storage. The decrease in proton transport coincided with losses in membrane phosphatidylcholine and was not associated with a change in vanadate-sensitive ATP hydrolysis. A technique based on a protocol developed for the reconstitution of Neurospora crassa plasma membrane H⁺-ATPase (DS Perlin, K Kasamo, RJ Brooker, CW Slayman 1984 J Biol Chem 259: 7884-7892) was employed to restore proton transport activity to maize microsomes. These results indicated that the decline in proton transport by maize root membranes during cold storage was not due to degradation of the protein moiety of the H⁺-ATPase, but was due to the loss of phospholipids.     

Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

The H⁺/ATP stoichiometry was determined for the plasma membrane H⁺-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H⁺/ATP stoichiometry utilized a kinetic approach where rates of H⁺ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H⁺ influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H⁺ leakage from a steadystate pH gradient after stopping the H⁺-ATPase or utilizing a mathematical model which describes the net transport of H⁺ at any given point in time. When the rate of H⁺ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H⁺/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (ΔG_atp), this value for H⁺/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Chilling-Induced Inactivation and Its Recovery of Tonoplast H-ATPase in Mung Bean Cell Suspension Cultures

The processes involved in adaptation to cold temperature were examined by growing suspension cultured cells of mung bean (Vigna radiata [L.] Wilczek) at 2°C for various periods of time and assaying the activities of various membrane-bound enzymes in vitro. The tonoplast H⁺-ATPase activity and the ATP-proton transport extracted from cells incubated at 2°C declined rapidly and reached a minimum level after 10 hours. The inactivation was reversible within 24 hours of chilling. The recovery of the cold-inactivated H⁺-ATPase was found to proceed in two steps, a faster recovery of ATP hydrolysis activity and a slower recovery of the proton transport. The recovery was markedly inhibited by the presence of azide, but not affected by 0.578 millimolar cycloheximide. This suggested the involvement of an energy process that had no requirement for de novo synthesis of protein. The cold-induced inactivation of the H⁺-ATPase may be due to a structural alteration of the enzyme. The slower recovery of proton transport relative to ATP hydrolysis during warming suggests that the protogenic domains in the enzyme may be affected differently by chilling.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.