Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H⁺-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent V_max for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent K_m for ATP was approximately 50 μM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (all inhibitors of vacuolar-type H⁺-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca²⁺. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-γ-³²P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H⁺-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H⁺-ATPase.     

Presence of 5-hydroxytryptamine (serotonin) transport coupled with vacuolar-type H(+)-ATPase in neurosecretory granules from bovine posterior pituitary

Electrogenic H(+)-ATPase was found in neurosecretory granules from bovine posterior pituitary. This enzyme was sensitive to bafilomycin, a specific inhibitor of vacuolar H(+)-ATPase, and was inactivated completely by cold treatment in the presence of MgATP and NaNO3. Immunoblot analysis showed the presence of immunologically identical polypeptides (72, 57, and 34 kDa) in the ATPases of the neurosecretory granules and chromaffin granules. The granules showed MgATP-dependent activity for 5-hydroxytryptamine (serotonin) uptake. This uptake was temperature-dependent and showed saturation kinetics (apparent Km for 5-hydroxytryptamine, 2 microM) and counter-flow. Reserpine and tetrabenazine at 1 microM inhibited the uptake, whereas imipramine at 2 microM had no effect. Dopamine, epinephrine and norepinephrine were also inhibitory. The uptake was abolished by various treatments that dissipated the electrochemical H+ gradient or inhibited the H(+)-ATPase. These results indicate that a vacuolar type H(+)-ATPase in the neurosecretory granules forms an electrochemical H+ gradient that drives 5-hydroxytryptamine uptake by a specific transport system. A similar granular fraction from the anterior pituitary had no ATP-dependent activity for 5-hydroxytryptamine uptake.     

Biochemical characterization of the yeast vacuolar H(+)-ATPase

The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) was repeated. This procedure consisted of isolation of vacuoles, solubilization with the zwitterionic detergent ZW3-14, and glycerol gradient centrifugation of the solubilized vacuoles. The fraction with the highest specific activity (11 mumol of ATP hydrolyzed mg-1 min-1) included eight polypeptides of apparent molecular masses of 100, 69, 60, 42, 36, 32, 27, and 17 kDa, suggesting that the enzyme may be more complex than the three-subunit composition proposed from the original purification. The 69-kDa polypeptide was recognized by antisera against the catalytic subunits of two other vacuolar ATPases and labeled with the ATP analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, indicating that it contains all or part of the catalytic site. A monoclonal antibody was prepared against this subunit. Under nondenaturing conditions, the antibody immunoprecipitated eight polypeptides, of the same molecular masses as those seen in the glycerol gradient fraction, from solubilized vacuolar vesicles. All eight of these polypeptides are therefore good candidates for being genuine subunits of the enzyme. The structure and function of the yeast vacuolar H+-ATPase were further characterized by examining the inhibition of ATPase activity by KNO3. In the presence of 5 mM MgATP, 100 mM KNO3 inhibited 71% of the ATPase activity of vacuolar vesicles, and the 69- and 60-kDa subunits (and possibly the 42-kDa subunit) were removed from the vacuolar membrane to a similar extent. At concentrations of less than 200 mM KNO3, the stripping of the ATPase subunits and the inhibition of ATPase activity were dependent on the presence of MgATP, suggesting that this is a conformation-specific disassembly of the enzyme. The yeast vacuolar H+-ATPase is a multisubunit enzyme, consisting of a combination of peripheral and integral membrane subunits. Its structure and subunit composition are very similar to other vacuolar ATPase, and it shares some characteristics with the F1F0-ATPases.     

The basal Mg2(+)-dependent ATPase activity is not part of the (H(+)+K+)-transporting ATPase reaction cycle.

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the 'stretched-out' version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.     

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.     

Identification and partial purification of a cytosolic activator of vacuolar H(+)-ATPases from mammalian kidney.

A factor that activates affinity-purified vacuolar H(+)-ATPase from bovine kidney microsomes was identified and partially purified from bovine kidney cytosol. The activator is a heat-stable, trypsin-sensitive acidic protein with a Mr by gel filtration of approximately 35,000. The activator increased the activity of renal microsomal and brush border H(+)-ATPase by over 60% but stimulated lysosomal H(+)-ATPase activity by only 28%; it had little or no activity against the remaining N-ethylmaleimide-insensitive ATPase in kidney microsomes and other transport ATPases. Stimulation of ATPase activity appeared to result from binding of the activator to the H(+)-ATPase. Activation was saturable, with a Hill coefficient of 1 at low protein concentrations. Both activator binding and stimulation of H(+)-ATPase activity were enhanced at pH values less than or equal to 6.5. The activator has selective effects on different H(+)-ATPases and is poised to activate the enzyme at low physiologic values of cytosolic pH; this newly identified cytosolic proteins may participate in the physiologic regulation of the vacuolar H(+)-ATPase.     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

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

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

Cold inactivation of vacuolar proton-ATPases

Incubation of the reconstituted H+-ATPase from chromaffin granules on ice resulted in inactivation of the proton-pumping and ATPase activities of the enzyme. Inactivation was dependent on the presence of Mg2+, Cl-, and ATP during the incubation at low temperature. Approximately 1 mM ATP, 1 mM Mg2+, and 200 mM Cl- were required for maximum inactivation. Incubation for about 10 min on ice was required to achieve 50% inactivation. A much smaller decline in activity was observed when the enzyme was incubated at room temperature with the same chemicals. Inactivation in the cold resulted in the release of five polypeptides from the membrane with apparent molecular masses of 72, 57, 41, 34, and 33 kDa on sodium dodecyl sulfate gels. Three of the polypeptides of 72, 57, and 34 kDa were identified as subunits of vacuolar H+-ATPases by antibody cross-reactivity. Similar results were obtained with several other vacuolar H+-ATPases including those from plant sources. It was concluded that the catalytic sector of the enzyme is released from the H+-ATPase complex by cold treatment, resulting in inactivation of the enzyme.     

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

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

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.     

Regulation and reversibility of vacuolar H(+)-ATPase

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

The mother-cell-membrane adenosine triphosphatase of sporulating Clostridium pasteurianum

1. Sporulation of Clostridium pasteurianum effects several changes in its proton-translocating cell-membrane H(+)-ATPase. Notable among these are the acquisition of susceptibility to activation by trypsin and a changed protein subunit composition. 2. A protein was isolated from the mother-cell membrane that inhibited the ATP phosphohydrolase activity of purified vegetative-cell-membrane H(+)-ATPase [BF(0)F(1) complex, which consists of soluble ATPase (BF(1)) and the proton-channel component (BF(0))] and rendered it susceptible to trypsin activation. 3. This trypsin-sensitive inhibitor protein had a molecular weight of 10000 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was indistinguishable from the novel protein subunit e of the mother-cell-membrane ATPase 4. In bacteriorhodopsin-containing everted membrane vesicles, the specific ATP synthetase activity of the mother-cell-membrane ATPase was significantly greater than that of the vegetative-cell-membrane ATPase. 5. Treatment with trypsin-sensitive inhibitor protein of artificial proteoliposomes containing bacteriorhodopsin and vegetative-cell-membrane H(+)-ATPase (BF(0)F(1)) significantly increased the specific ATP synthetase activity of this enzyme. 6. The ATP synthetase activity of crude cell-membrane preparations from cultures of Clostridium pasteurianum increased during that period in the course of sporulation when the membrane ATP phosphohydrolase was both most rapidly decreasing in specific activity and acquiring its susceptibility to activation by trypsin.     

Degradation of pyrene by indigenous fungi from a former gasworks site

Salt tolerance in Saccharomyces cerevisiae is a complex trait, involving regulation of membrane polarization, Na(+) efflux and sequestration of Na(+) in the vacuole. Since transmembrane transport energized by H(+)-adenosine triphosphatases (ATPases) is common to all of these tolerance mechanisms, the objective of this study was to characterize the responses of the plasma membrane H(+)-ATPase, vacuolar H(+)-ATPase and mitochondrial F(1)F(0)-ATPase to NaCl stress. We hypothesized that since the vacuolar ATPase is responsible for generating the proton motive force required for import of cations (such as Na(+)) into the vacuole, strains lacking this activity should be hypersensitive to NaCl. We found that strains lacking vacuolar ATPase activity were in fact hypersensitive to NaCl, while strains lacking ATP synthase were not. This effect was specific to the ionic component of NaCl stress, since the mutant strains were indistinguishable from wild-type and complemented strains in the presence of sorbitol.     

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.     

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.     

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora

Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.