大豆液泡膜H~+-ATPase的纯化和重组

通过不连续蔗糖密度梯度离心得到的液泡膜微囊 ,先由胆酸钠和 OG分步破膜抽提、经阴离子交换柱 ( Q- Sepharose)层析分离 .纯化后的酶含 V型 H+ - ATPase的主要亚基 ,与大豆磷脂重组 ,获得了有较高泵活性的脂酶体 .脂酶体的质子泵活性受 Valinomycin激活 ,说明它是致电性的 ,受NO-3 ,DCCD以及特异性的 V型 ATPase抑制剂 Bafilomycin的抑制 .脂酶体的泵活性不受 F型和P型 ATPase抑制剂抑制 ,表明质子转运是由 V型 H+ - ATPase引起的 .     

Rapid purification and reconstitution of a plant vacuolar ATPase using Triton X-114 fractionation: subunit composition and substrate kinetics of the H(+)-ATPase from the tonoplast of Kalancho? daigremontiana.

A rapid procedure for the purification and reconstitution into proteoliposomes of the H(+)-translocating ATPase of plant vacuolar membranes is reported. It involves fractionation of the tonoplast with Triton X-114, resolubilization of the ATPase with octyl glucoside in the presence of a mixture of phosphatidylcholine, phosphatidylserine and cholesterol (27:53:20, by weight), and removal of the detergent by gel-filtration. Starting with partially purified vacuolar membranes, the procedure can be accomplished in about 2 hours. It has been applied to the H(+)-ATPase from the crassulacean plant Kalancho? daigremontiana, from which it yields vesicles with a specific ATPase activity of about 3 mumol/min per mg protein. The purified enzyme contains polypeptides of apparent molecular mass 72, 57, 48, 42, 39, 33 and 16 kDa; these polypeptides also co-sediment on centrifugation of the solubilized ATPase through glycerol gradients. The 16-kDa subunit is labelled with [14C]dicyclohexylcarbodiimide. There is no evidence for a larger ATPase subunit in this preparation. The reconstituted ATPase proteoliposomes undergo ATP-dependent acidification, which can be measured by quenching of the fluorescence of 9-aminoacridine. The initial rate of fluorescence quenching is a measure of the rate of H+ translocation, and is directly proportional to the vesicle protein concentration, so the preparation is suitable for studying the kinetics of the tonoplast H(+)-ATPase. The dependence of the rate of fluorescence quenching on the concentration of MgATP is well fitted by the Michaelis equation, with a Km value about 30 microM. ATP can be replaced by dATP, ITP, GTP, UTP or CTP, and Mg2+ by Mn2+ or Ca2+; kinetic parameters for these substrates are reported. In contrast, hydrolysis of MgATP shows complex kinetics, suggestive either of negative cooperativity between nucleotide-binding sites, or of two non-interacting catalytic sites. Both the hydrolytic and the H(+)-translocating activities of the proteoliposomes are inhibited by nitrate, though not in parallel, the latter activity being the more sensitive. Both activities are inhibited in parallel by bafilomycin A1, which does not produce complete inhibition; the bafilomycin-insensitive component has complex ATPase kinetics similar to those of the uninhibited enzyme.     

Solubilization of a phospholipid-stimulated adenosine triphosphatase complex from membranes of Escherichia coli.

A phospholipid-stimulated adenosine triphosphatase (ATPase) complex was solubilized and partially purified from membrane particles of Escherichia coli ML308-225. The complex was of large molecular size and contained 16 polypeptides, five of which were subunits of the F₁-type ATPase of E. coli. Components of the respiratory chain were absent. Enzyme activity was stimulated by lysophosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, and cardiolipin but not by phosphatidylethanolamine. The ATPase activity of the complex was inhibited by N,N′-dicyclohexylcarbodiimide and by Dio-9 at lower inhibitor:protein ratios than required for inhibition of the F₁-type ATPase of E. coli. However, the ATPase complex was less sensitive than the membrane-bound enzyme to inhibition by these compounds.     

The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium Clostridium pasteurianum. 1. ATP phosphohydrolase activity.

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.     

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.     

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).     

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.     

Immunoaffinity purification and characterization of vacuolar H+ATPase from bovine kidney

Vacuolar proton-translocating ATPase from bovine kidney was purified in one step by immunoprecipitation and immunoaffinity chromatography using an immobilized anti-H+ATPase monoclonal antibody. The monoclonal antibody affinity matrix coprecipitated polypeptides with Mr of 70,000, a cluster at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000 from solubilized bovine kidney microsomal membranes, a pattern that was unaffected by different detergent washing conditions. A nearly identical pattern of polypeptides was observed in H+ATPase partially purified by an entirely independent method. The immunoaffinity purified H+ATPase had reconstitutively active ATP-induced acidification and potential generation that was inhibited by N-ethylamaleimide. The purified enzyme had specific activities as high as 3.1 mumol/min/mg protein, dual pH optima at 6.5 and 7.2, and a Km for ATP of 150 microM. The substrate preference was ATP greater than ITP much greater than UTP greater than GTP greater than CTP. The affinity purified H+ATPase was stimulated by phosphatidyl glycerol greater than phosphatidyl inositol much greater than phosphatidyl choline greater than phosphatidyl serine. The immunoaffinity purified enzyme did not require monovalent anions or cations for activity, and the divalent cation preference for activity was Mn, Mg much greater than Ca greater than Co much greater than Sr, Ba. The enzyme was not inhibited by ouabain, azide, or vanadate, but had kappa 1/2 inhibitory concentrations of 22.2 microM for N-ethylmaleimide, 14.9 microM for NBD-Cl, 4.9 microM for N,N'-dicyclohexylcarbodiimide, 13.8 microM for 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and 315 microM for Zn, values close to those for inhibition of proton transport in the native vesicles. The affinity purified kidney enzyme has similarities to but also significant differences in structural and enzymatic properties from those reported for other vacuolar H+ATPase.     

Solubilization by lysolecithin and purification of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe.

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient. The specific activity of the Mg2+-requiring plasma membrane ATPase activity (EC 3.6.1.3) was enriched from 0.3 mumol min-1 x mg-1 of protein in the homogenate to 26 in the purified membranes. The optimal conditions for solubilization of the ATPase activity by lysolecithin were found to be: 2 mg/ml of lysolecithin, a lysolecithin to protein ratio of 8 at pH 7.5, and 15 degrees C in the presence of 1 mM ATP and 1 mM ethylenediaminetetraacetic acid. A 6- to 7-fold purification of the solubilized ATPase activity was obtained by centrifugation of the lysolecithin extract in sucrose gradient. Part of the ATPase activity which was inactivated during the centrifugation in the sucrose gradient could be restored by addition of a micellar solution of 50 microgram of lysolecithin/ml during the assay. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purified enzyme showed only one band of Mr = 105,000 stained with Coomassie blue. Another ATPase component of apparent molecular weight lower than 10,000 was stained by periodic Schiff reagent but not colored by Coomassie blue. The purified enzyme was 85% inhibited by 50 micrometer N,N'-dicyclohexylcarbodiimide and 94% inhibited by 53 microgram of Dio-9/ml.     

Partial purification and characterization of an anion-activated ATPase from radish microsomes

Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.     

Properties of the partially purified tonoplast H+-pumping ATPase from oat roots

Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast ATPase preparation. Two major polypeptides, 72 and 60 kDa, that copurified with the ATPase activity and the 14-18-kDa DCCD-binding peptide are postulated to be subunits of a holoenzyme of 300-600 kDa (estimated by gel filtration). Despite several catalytic similarities with the mitochondrial H+-ATPase, the major polypeptides of the tonoplast ATPase differed in mass from the alpha and beta subunits (58 and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat F1F0-ATPase.     

Characterization of a Dio-9-resistant strain of Saccharomyces cerevisiae.

The ATPase inhibitor Dio-9 effectively suppressed a number of physiological processes in a wild-type strain of Saccharomyces cerevisiae, X2180-1A. Low levels of the antibiotic inhibited cell growth, amino acid transport, hydrogen ion efflux, and ATPase activity. In addition, Dio-9 acted as a permeabilizing agent for the yeast plasma membrane. A mutant yeast strain, XC24, was selected on the basis of its ability to grow on minimal medium containing 200 μg/ml of Dio-9. Strain XC24 had acquired a pH-conditional ability to resist the permeabilizing effects of Dio-9. In addition, amino acid transport and hydrogen ion pumping exhibited a reduced senstivity to Dio-9 at low pH in the mutant strain. Strain XC24 was also resistant to the permeabilizing effects of the basic polymers protamine and deacylated chitin.     

Isolation and reconstitution of the chloride transporter of clathrin-coated vesicles

Clathrin-coated vesicle acidification is mediated by an endomembrane proton translocating ATPase. This pump is electrogenic, and significant pH gradient formation requires the parallel movement of chloride through a chloride transporter in order to maintain net electroneutrality. We have solubilized, isolated and achieved 270-fold purification of this chloride transporter by means of selective detergent solubilization with cholate and polyoxyethelene 9-lauryl ether (C12E9), hydroxylapatite chromatography, and glycerol gradient centrifugation. Stabilization of the solubilized transporter requires 5 mM dithiothreitol. The partially purified transporter was co-reconstituted with the purified clathrin-coated vesicle proton translocating complex to yield preparations of proteoliposomes capable of valinomycin-independent proton pumping, as assessed by ATP-generated acridine orange quenching. In addition, the chloride transporter was independently reconstituted and was shown to catalyze diisothiocyano-disulfonic acid stilbene-sensitive 36Cl uptake. The anionic conductive selectivity of the reconstituted transporter (chloride = bromide greater than nitrate) exactly matched that of the transporter of native clathrin-coated vesicles. These studies demonstrate that the chloride transporter of vacuolar acidification systems is structurally and functionally dissociable from co-existing proton pumps and allow for investigations of pump-transporter interactions in a resolved system.     

Stimulatory effect of insecticides on partially purified P-glycoprotein ATPase from the resistant pest Helicoverpa armigera.

A P-glycoprotein-like protein (Ha-Pgp) was detected in a membrane preparation from the insecticide-resistant pest Helicoverpa armigera (Lepidoptera: Noctüidae) using C219 antibodies that are directed towards an epitope in the nucleotide-binding domains. This protein was partially purified and found to be a glycoprotein displaying ATPase activity. SDS-PAGE confirmed that a high molecular mass glycoprotein (150 kDa) was overexpressed in resistant pests, but was not detected in susceptible pests. The partially purified Ha-Pgp ATPase was reconstituted into proteoliposomes and it was found that some insecticides, namely, monocrotophos, endosulfan, cypermethrin, fenvalerate, and methylparathion, stimulated the ATPase activity. The effect of various inhibitors on partially purified Ha-Pgp showed that orthovanadate is a potent inhibitor of its ATPase activity, inhibiting it by 90% at a concentration of 2 mmol/L. Other inhibitors, such as EDTA, sodium azide, and molybdate resulted in only a 20% decrease in activity. Details of the structure and function of Ha-Pgp will be important in the development of strategies to overcome insecticide resistance in this pest.     

Subunit composition and Ca(2+)-ATPase activity of the vacuolar ATPase from barley roots.

The vacuolar ATPase was purified from a tonoplast-enriched membrane fraction from barley (Hordeum vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KNO3 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purified holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca(2+)-ATPase were obtained from the Sephacryl S-400 column. A peak of Ca(2+)-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO3- and DCCI. It is proposed that this Ca(2+)-ATPase is a partial reaction of the plant vacuolar ATPase. The second Ca(2+)-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO3- or DCCI. The second Ca(2+)-ATPase is a major component of ATP hydrolysis by the native membranes.     

Production and characterization of a monoclonal antibody to vacuolar H+ATPase of renal epithelia

A monoclonal antibody to vacuolar H+ATPase isolated from bovine kidney medulla was produced and characterized by immunoprecipitation and immunocytochemistry. The antibody, immobilized on beads, specifically immunoprecipitated both solubilized N-ethylmaleimide-sensitive ATPase activity and proton-transporting vesicles from renal microsomes; control experiments with an "irrelevant" monoclonal antibody showed no immunoprecipitated activity. By fluorescent immunocytochemistry, the antibody stained the membranes of intracellular vacuolar compartments in LLC-PK1 cells. Immunocytochemical staining showed that the monoclonal antibody colocalized partially with N-(3-[2,4-dinitrophenyl)amino)propyl)-N-(3-amino-propyl)methylamine, a probe for acidic compartments, with the endocytic markers dextran and transferrin, with the lysosomal probe alpha 2-macroglobulin, and with clathrin. The anti-vacuolar H+ATPase antibody showed no colocalization with staining for mitochondrial H+ATPase. The anti-vacuolar H+ATPase antibody should serve as a specific probe for examining the distribution and dynamics of the vacuolar proton pump in renal epithelial cells.     

Potassium transport coupled to ATP hydrolysis in reconstituted proteoliposomes of yeast plasma membrane ATPase

Potassium transport coupled to ATP hydrolysis has been reconstituted in proteoliposomes using a highly purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe. The ATPase activity in the incorporated enzyme was strongly stimulated (2.2-fold) by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP). The H+/K+ exchanger nigericin (in the presence of K+) stimulated 1.6-fold the ATPase activity. When both ionophores were added together, the stimulation was increased up to 2.7-fold. When a potassium concentration gradient (high K+ in) was applied to the proteoliposome membrane, a significant drop in the CCCP-stimulated ATPase activity was observed. Inversion of the K+ concentration gradient (high K+ out) did not decrease the stimulation by CCCP. High Na+ in also decreased the stimulation induced by CCCP in the absence but not in the presence of external K+. However, high Li+ in had no effect. Direct potassium efflux from the proteolyposomes was detected upon addition of MgATP using a selective K+ electrode. The ATP-dependent potassium efflux was abolished in CCCP and/or nigericin-pretreated proteoliposomes. However, during steady state ATP hydrolysis, a transient and small K+ efflux was observed upon addition of a CCCP pulse. I propose that the plasma membrane Mg2+-dependent ATPase in yeast cells not only carries out electrogenic H+ ejection but also drives the uptake of potassium via a voltage-sensitive gate which is closed in the absence and open in the presence of the membrane potential.     

Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.