Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.     

Acidocalcisomes are functionally linked to the contractile vacuole of Dictyostelium discoideum.

The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H(+)-ATPase, an H(+)-pyrophosphatase, and a Ca(2+)-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H(+)-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4' -diisothiocyanatodihydrostilbene-2, 2' -disulfonic acid and the vacuolar H(+)-ATPase inhibitor bafilomycin A(1). Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.     

Characterization of a Vacuolar Pyrophosphatase in Trypanosoma brucei and Its Localization to Acidocalcisomes

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH(4)Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H(+)-pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 microM) and unaffected by bafilomycin A(1) (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 microM), oligomycin (1 microM), N-ethylmaleimide (100 microM), and KNO(3). AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H(+)-ATPase, H(+)-PPase, and (ADP-dependent) H(+)/Na(+) antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H(+)-PPase (both stages) and H(+)/Na(+) exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles.     

A calcium pump in plasma membrane vesicles from Leishmania braziliensis

A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.     

Isolation and properties of bovine kidney brush border vacuolar H(+)-ATPase. A proton pump with enzymatic and structural differences from kidney microsomal H(+)-ATPase

Vacuolar H(+)-ATPase was isolated from highly purified bovine kidney brush border, using a previously described immunoaffinity method. The affinity purified enzyme had reconstitutively active ATP-induced acidification that was inhibited by N-ethylmaleimide. The brush border H(+)-ATPase had a single pH optimum of 7.3, and a single Km for ATP of 360 microM. The enzyme showed no lipid activation; it had a substrate preference of ATP greater than ITP greater than UTP greater than GTP much greater than CTP, with an ATP:GTP selectivity of 1.69. The brush border H(+)-ATPase required no monovalent anion or cation for activity and was inhibited by the oxyanions NO3(-1) much greater than SO4(-2); sulfite stimulated activity at low concentrations and inhibited at higher concentrations. The inhibition produced by nitrate could not be attributed to dissociation of subunits from the enzyme. The divalent or trivalent cation preference was Mn+2 much greater than Mg+2 much greater than Co+2 greater than Al+3 greater than Ca+2 much greater than Ba+2,Sr+2; 1 mM Zn+2 inhibited the enzyme completely, but Cu+2 inhibited only 49% of activity at concentrations up to 5 mM. Sodium dodecyl sulfate-polyacrylamide gels of the brush border H(+)-ATPase showed subunits at Mr 70,000, a doublet at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000. On two-dimensional gels, the pl value for the Mr 70,000 subunit was 6.3, for the Mr 56,000 was 6.4, and for the Mr 31,000 was 7.5-8.5, and microheterogeneity was observed in the Mr 56,000 and 31,000 subunits. A comparison of kidney cortex brush border H(+)-ATPase with kidney cortex microsomal H(+)-ATPase revealed differences in pH optimum, Km for ATP, lipid dependence, substrate preference, divalent ion preference, copper sensitivity, and in microheterogeneity of the Mr 56,000 and 31,000 subunits, providing evidence that different functional and structural classes of vacuolar H(+)-ATPase are segregated to specific membrane compartments.     

Mechanism of stimulation of Ca2+ uptake by miconazole and ethidium bromide in yeasts: role of vacuoles in Ca2+ detoxification

The antifungal antibiotic miconazole and the cationic dye ethidium bromide, both caused K+ efflux, membrane depolarization and intracellular acidification in the yeast Saccharomyces cerevisiae. Whereas miconazole inhibited the activity of the plasma membrane H+-ATPase, no such inhibition was observed using ethidium bromide at concentrations up to 600 microM. Low concentrations of both drugs caused marked stimulation of the energy dependent Ca2+ uptake. The extra Ca2+ taken up in the presence of the drugs was localized within the vacuoles, whereas K+ was lost mainly from the cytosolic pool. The ions Zn2+ and La3+ inhibited the effect of both drugs on the stimulation of Ca2+ uptake. The results indicated that both drugs caused an increase in the permeability of cell membranes to ions, leading to an increase in the influx of Ca2+ into the cytosol along its electrochemical gradient. Consequently, the concentration of Ca2+ within the cytosol increased and in turn led to the enhancement of Ca2+ uptake by the energy dependent vacuolar Ca2+ system, which functioned as a Ca2+ detoxification system.     

Characterization of a vacuolar proton ATPase in Dictyostelium discoideum

Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.     

Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.     

Partial Purification and Characterization of the Vacuolar H+-ATPase of Mammalian Synaptic Vesicles

Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H(+)-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H(+)-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute approximately 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H(+)-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of approximately 1.7 nmol/mg of protein. These findings indicate that the vacuolar H(+)-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.     

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.     

Guanine derivatives modulate L-glutamate uptake into rat brain synaptic vesicles

Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.     

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.     

Induction of high affinity phosphate transporter in the duckweed Spirodela oligorrhiza

Duckweed plants (Spirodela oligorrhiza) grown under phosphate (Pi)-deficient conditions (- P plants) exhibited more than 50-fold higher Pi uptake activity than plants grown under Pi-sufficient conditions (+ P plants). The Pi uptake activity of - P plants measured using (32)Pi was significantly inhibited by carbonylcyanide m-chlorophenylhydrazone, indicating that Pi uptake is energized by the electrochemical proton gradient across the plasma membrane (PM). When Pi uptake was examined at various concentrations of Pi, more active uptake of Pi was observed in - P plants than in + P plants, irrespective of the Pi concentrations. An immunoblot analysis of the PM proteins using antiserum against the conserved sequence of the high-affinity Pi transporter recognized the occurrence and large accumulation of a novel protein band at 48 kDa in - P plants. The protein was almost completely extracted with chloroform-methanol (2:1, v/v), but only a trace amount of the protein was detected in + P plants. Immunohistochemical studies of plant roots using the same antiserum demonstrated a large accumulation of high-affinity Pi transporters at the outermost cortical cells of - P plants, but not of + P plants. When an immunoblot analysis of PM proteins was performed using antiserum against the PM H(+)-ATPase, a positive band of about 96 kDa was detected in both plants with a similar signal intensity. Furthermore, ATP-hydrolytic and ATP-dependent H(+)-transporting activities of PM H(+)-ATPase in - P plants were not higher than those in + P plants. However, kinetic analyses showed that the PM H(+)-ATPase in - P plants had a lower K(m) value and a higher coupling efficiency between ATP hydrolysis and H(+) pumping than the corresponding values in + P plants. These results suggest that the significant stimulation of Pi uptake in - P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in the PM, and that the electrochemical proton gradient across the PM may be generated by the high-ATP-affinity and energy-efficient H(+) pump in - P plants. This would facilitate the acquisition of Pi in S. oligorrhiza under Pi-depleted conditions.     

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 Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 microM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (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 (-gamma-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.     

Proton potential-dependent polyamine transport by vacuolar membrane vesicles of Saccharomyces cerevisiae.

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulated spermine and spermidine in the presence of ATP, not in the presence of ADP. Spermine and spermidine transport at pH 7.4 showed saturation kinetics with Km values of 0.2 mM and 0.7 mM, respectively. Spermine uptake was competitively inhibited by spermidine and putrescine, but was not affected by seven amino acids, substrates of active transport systems of vacuolar membrane. Spermine transport was inhibited by the H(+)-ATPase-specific inhibitors bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, but not by vanadate. It was also sensitive to Cu2+ or Zn2+ ions, inhibitors of vacuolar H(+)-ATPase. Both 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847) and nigericin blocked completely the spermine uptake, but valinomycin did not. [14C]Spermine accumulated in the vesicles was exchangeable with unlabeled spermine and spermidine. However, it was released by a protonophore only in the presence of a counterion such as Ca2+. These results indicate that a polyamine-specific transport system depending on a proton potential functions in the vacuolar membrane of this organism.     

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.     

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.     

The HPV-16 E5 oncogene and bafilomycin A(1) influence cell motility

It is known that the proper function of the vacuolar H(+)-ATPase is inhibited by bafilomycin A(1). In transfected cells the E5 protein interacts with the 16 kDa subunit of the vacuolar H(+)-ATPase. Thereby the pH gradient in endocytic structures is impaired. The present study demonstrates for the first time that the inhibition of the vacuolar H(+)-ATPase in NIH3T3 cells with bafilomycin A(1) or by transfection of cells with the HPV-16 E5 oncogene leads to a changed morphology and a reduced motility as shown by computer-assisted video recordings and image analysis. Bafilomycin A(1) potentiates the effect of the E5 protein on cell motility and this cooperative effect indicates that the E5 protein and bafilomycin A(1) either target the vacuolar H(+)-ATPase differently or that the E5 protein has additional targets in transfected cells. Our data therefore show that proper function of the vacuolar H(+)-ATPase is needed for normal cell locomotion.     

Characterization of isolated acidocalcisomes of Trypanosoma cruzi

The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000 x g) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca(2+) uptake (Ca(2+)-ATPase) activity was detectable in the isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified, and the rate of Ca(2+) uptake increased. Use of the indicator Oxonol VI showed that V-H(+)-PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H(+)-PPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H(+)-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H(+)-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H(+)-ATPase and V-H(+)-PPase activities are present in the same Ca(2+)-containing compartment.     

H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.