Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The function of the Mg(2+)-requiring KCl-stimulated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of higher plants in active ion transport was investigated by using a purified microsomal fraction containing sealed plasma membrane vesicles. (Sze, H. (1980) Proc. Natl. Acad. Sci. USA 77, 5904-5908). A transmembrane electrical potential (+30 to +44 mV), monitored by uptake of a permeant anion ((35)SCN(-)), was generated specifically by ATP in purified microsomal vesicles of tobacco callus. ATP-dependent (35)SCN(-) uptake required Mg(2+), was optimal at pH 6.75, and showed similar ATP concentration dependence as the Mg(2+)-requiring KCl-stimulated ATPase activity. Plasma membrane ATPase inhibitors (N,N'-dicyclohexylcarbodiimide and vanadate) inhibited generation of the ATP-dependent electrical potential. A proton conductor (carbonyl cyanide m-chlorophenylhydrazone), but not a K(+) ionophore (valinomycin), completely collapsed the electrical potential. The results provide in vitro evidence that the Mg(2+)/KCl-ATPase of higher plants is an electrogenic pump. These results are consistent with the hypothesis that an electrogenic H(+) pump is catalyzed by the plasma membrane ATPase of plants.
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PMID:Mg/KCl-ATPase of plant plasma membranes is an electrogenic pump. 1659 89

Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [(14)C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [(14)C]SCN(-) uptake and (36)Cl(-) uptake activities coincided with [(14)C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker.
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PMID:Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation. 1666 55

When assayed in the presence of azide, NO(3) (-) was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 x M017) root microsomal membranes. The distribution of the NO(3) (-)-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H(+)-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 mum vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO(3) (-)-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H(+) from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K(+) > NH(4) (+) > Rb(+) > Cs(+) > Li(+) > Na(+) > choline(+)) whereas the NO(3) (-)-sensitive ATPase was stimulated by anions (Cl(-) > Br(-) > C(2)H(3)O(2) (-) > SO(4) (2-) > I(-) > HCO(3) (-) > SCN(-)). Both ATPases required divalent cations. However, the order of preference for the NO(3) (-)-sensitive ATPase (Mn(2+) > Mg(2+) > Co(2+) > Ca(2+) > Zn(2+)) differed from that of the vanadate-sensitive ATPase (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO(3) (-)-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO(3) (-)-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO(3) (-)-sensitive ATPase was stimulated by gramicidin and was associated with NO(3) (-)-inhibitable H(+) transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO(3) (-)-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO(3) (-) sensitivity is a better criterion by which to identify this ATPase than either Cl(-) stimulation or H(+) transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.
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PMID:Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots. 1666 96

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H(+)/K(+) exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN(-) distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl(-) and inhibited by NO(-) (3) or 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The distribution of Cl(-)-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H(+) pumping into vesicles as monitored by [(14)C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K(+). These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl(-)-stimulated, H(+)-pumping ATPase may be enriched in vacuolar-type membranes; the H(+)-pumping ATPase that is stimulated by K(+) and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.
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PMID:Separation of two types of electrogenic h-pumping ATPases from oat roots. 1666 44

The H(+)-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl(-) and NO(3) (-). N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K(m) of the enzyme for ATP but caused relatively little alteration of the V(max). Nitrate increased K(m) only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl(-). Other monovalent anions that specifically increased the K(m) of the ATPase for ATP were, in order of increasing K(i), SCN(-), ClO(4) (-), and ClO(3) (-). Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO(3) (-), was a competitive inhibitor of the ATPase but ADP and NO(3) (-) did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO(3) (-) does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg.ADP and Mg.ATP protected the enzyme from inactivation by NEM but Mg.ADP was the more potent of the two. Chloride and NO(3) (-) exerted little or no effect on the protective actions of Mg.ADP and Mg.ATP suggesting that neither Cl(-) nor NO(3) (-) are involved in substrate binding.
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PMID:Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate. 1666 60

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Deltapsi) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H(+)-pumping pyrophosphatase, which is K(+) stimulated and anion insensitive. The relative rate of DeltaPsi dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN(-) (100) > NO(3) (-) (72) = Cl(-) (70) > Br(-) (62) > SO(4) (2-) (5) = H(2)PO(4) (-) (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Deltapsi dissipation by Cl(-) and NO(3) (-), but not by SCN(-), was saturable. The K(m) values for Cl(-) and NO(3) (-) uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H(+)-coupled Cl(-) transporter on the same vesicles, the potential-dependent Cl(-) transport was insensitive to 4,4'-diisothiocyano-2,2'-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl(-) transport in these vesicles. The potentials generated by the H(+)-translocating ATPase and H(+)-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl(-) conductance on the anion-sensitive H(+)-ATPase was found.
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PMID:Potential-dependent anion transport in tonoplast vesicles from oat roots. 1666 76

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K(m) of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30 degrees C. Several effectors known to inhibit ATPase (N,N'-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H(+) electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN(-) and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH-, OH-, and NH(2)- groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.
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PMID:Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells. 1666 82

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the DeltapH probe [(14)C]methylamine. Evidence is presented for the operation of Na(+)/H(+) and K(+)/H(+) antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H(+)-ATPase or by imposing an artificial pH gradient) brings about dissipation of the DeltapH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl(-), using the DeltaPsi probe SCN(-). Cation/H(+) exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H(+)-ATPase; coupling of the opposed cation and H(+) fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na(+)/H(+) and K(+)/H(+) antiport in Atriplex. Moreover, additive effects are obtained when Na(+) is supplied together with saturating concentrations of K(+), and vice versa, suggesting that separate antiporters for Na(+) and for K(+) may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.
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PMID:Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton. 1666 18

We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substrate-specific, K(+)/H(+) antiport in both tonoplast and plasma membrane-enriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K(+) (with Cl(-) or SCN(-)) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H(+)-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K(+)/H(+) exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCl was supplied, even in the absence of 100 millimolar Cl(-) and, second, efflux of K(+) from K(+)-loaded vesicles drives intravesicular accumulation of H(+) against the electrochemical potential gradient. Neither was the exchange due to competition between K(+) and quinacrine for membrane sites, nor to inhibition of the H(+)-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K(+) (at micromolar concentrations); Na(+)/H(+) antiport was detected only at concentrations two orders of magnitude higher. Rb(+), Li(+), or Cs(+) were ineffective. Dependence of tonoplast K(+)/H(+) antiport on K(+) concentration was complex, showing saturation at 10 millimolar K(+) and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar NO(3) (-) and showed no sensitivity to vanadate or oligomycin) and with plasma membrane-enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol-sensitive but showed no sensitivity to NO(3) (-) or oligomycin). The possible physiological role of such a K(+)/H(+) exchange mechanism is discussed.
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PMID:Evidence for a highly specific k/h antiporter in membrane vesicles from oil-seed rape hypocotyls. 1666 11

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori, the bacterium associated to gastroduodenal ulcers and stomach cancers. VacA induces formation of cellular vacuoles that originate from late endosomal compartments. VacA forms an anion-selective channel and its activity has been suggested to increase the osmotic pressure in the lumen of these acidic compartments, driving their swelling to vacuoles. Here, we have tested this proposal on isolated endosomes that allow one to manipulate at will the medium. We have found that VacA enhances the v-ATPase proton pump activity and the acidification of isolated endosomes in a Cl- dependent manner. Other counter-anions such as pyruvate, Br-, I- and SCN- can be transported by VacA with stimulation of the v-ATPase. The VacA action on isolated endosomes is associated with their increase in size. Single amino acid substituted VacA with no channel-forming and vacuolating activity is unable to induce swelling of endosomes. These data provide a direct evidence that the transmembrane VacA channel mediates an influx of anions into endosomes that stimulates the electrogenic v-ATPase proton pump, leading to their osmotic swelling and transformation into vacuoles.
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PMID:The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes. 1725 77


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