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 basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of chloride channels in membrane vesicles from the kidney outer medulla. 246 22

The pathway(s) of passive conductive Cl transport across isolated frog skin are analyzed by electrophysiological techniques including microelectrode impalement of principal cells. It is found that the apical membrane of granular cells is virtually impermeable for Cl. Substitution of mucosal Cl by anions except NO3 and SCN decreases Cl-related tissue conductance (gCl) with first order kinetics. NO3 and SCN block gCl with half-maximal concentration of 18 and 5 mM, respectively. Omission of serosal Cl has little effect on gCl unless the inhibiting anions NO3 or SCN are used. The putative Cl channel blocker diphenylaminocarbonic acid (DPC) and some analogs inhibit gCl, half-inhibitory concentration of the most potent dichloro-DPC is 10(-6) M. The inhibitors act only from the mucosal side. Slow dissipation of gCl after abolition of Na entry across the apical membranes which can be prevented by preceding blockage of the Na-K-ATPase with ouabain suggests that the intracellular Na activity might influence the permeability of the Cl pathway. It is supposed that this control involves microfilaments between the cytoskeleton and the tight junctions with Cl-specific permeation sites in outer regions of the junctional complex.
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PMID:Chloride conductance of frog skin: localization to the tight junctions? 247 Oct 51

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.
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PMID:Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport. 256 4

ATP-driven proton transport in intact clathrin-coated vesicles requires the presence of a permeant anion, such as Cl-, to provide charge compensation during the electrogenic movement of protons. Using the purified (H+)-ATPase from clathrin-coated vesicles in both the detergent-solubilized and reconstituted states, we have studied the direct effects of anions on the activity of this enzyme. Both proton transport and ATP hydrolysis by the purified enzyme are independent of the presence of Cl-. In addition, proton transport does not occur even at high Cl- concentrations unless K+ and valinomycin are present to dissipate the membrane potential generated. These results indicate that the anion channel which provides for Cl- flux in intact coated vesicles is not a component of the purified (H+)-ATPase. Inhibition of ATPase activity is observed in the presence of I-, NO3-, or SO4(2-), with 50% inhibition occurring at 350 mM I-, 50 mM NO3-, or 40 mM SO4(2-). The presence of ATP lowers the concentration of I- required for 50% inhibition from 350 mM to 100 mM and increases the maximal inhibition observed in the presence of NO3- from 65% to 100%. Two separate mechanisms appear to be responsible for anion inhibition of the (H+)-ATPase. Thus, I- and high concentrations of NO3- (in the presence of ATP) cause inhibition by dissociation of the (H+)-ATPase complex, while SO4(2-) and NO3- (in the absence of ATP) cause inhibition without dissociation of the complex, suggesting the existence of an inhibitory anion binding site on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of anions and ATP with the coated vesicle proton pump. 256 25

The effect of Cl- and other anions on the tonoplast H+-translocating ATPase (H+-ATPase) from Hevea brasiliensis (rubber tree) latex was investigated. Cl- and other anions stimulated the ATPase activity of tightly sealed vesicles prepared from Hevea tonoplast, with the following decreasing order of effectiveness: Cl- greater than Br- greater than SO4(2-) greater than NO3-. As indicated by the changes of the protonmotive potential difference, anion stimulation of tonoplast H+-ATPase was caused in part by the ability of these anions to dissipate the electrical potential. This interpretation assumes not a channelling of these anions against a membrane potential, negative-inside, but a modification of the permeability of these ions through the tonoplast membrane. In addition, Cl- and the other anions stimulated the ATPase activity solubilized from the tonoplast membrane. Consequently, the tonoplast H+-pumping ATPase can be considered as an anion-stimulated enzyme. These results are discussed in relation to various models described in the literature for the microsomal H+-ATPase systems claimed as tonoplast entities.
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PMID:Chloride-ion stimulation of the tonoplast H+-translocating ATPase from Hevea brasiliensis (rubber tree) latex. A dual mechanism. 257 42

Thiamine triphosphatase (TTPase) from membranes isolated from the main electric organ of E. electricus is activated about 8 fold by NO3-, I- and SCN- while SO42- is inhibitory. Activating anions shift the pH optimum of the enzyme from 5.0 to 8.0. The enzyme is irreversibly inactivated by low concentrations of 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS), an inhibitor of anion transport. Anions protect from DIDS inactivation. These and other results suggest that the membrane-bound TTPase activity is tightly controlled, possibly through mechanisms involving anion transport.
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PMID:Thiamine triphosphatase from Electrophorus electric organ is anion-dependent and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'disulfonic acid. 284 36

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification. 285 93

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.
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PMID:Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution. 286 82

The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.
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PMID:Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange. 287 Oct 45

Purified goblet cell apical membranes from Manduca sexta larval midgut exhibit a specific ATPase activity approx. 20-fold higher than that in the 100 000 X g pellet of a midgut homogenate. The already substantial ATPase activity in this plasma membrane segment is doubled in the presence of 20-50 mM KCl. At ATP concentrations ranging from 0.1 to 3.0 mM, the presence of 20 mM KCl leads to a 10-fold increase in the enzyme's affinity for ATP. ATPase activity is greatest at a pH of approx. 8. In addition to ATP, GTP serves as a substrate, but CTP, ADP, AMP and p-nitrophenyl phosphate do not. Either Mg2+ or Mn2+ is required for activity and cannot be replaced by Ca2+ or Zn2+. The ATPase activity of goblet cell apical membranes is inhibited by neither the typical (Na+ + K+)-ATPase inhibitors, ouabain and orthovanadate, nor by the typical mitochondrial F1F0-ATPase inhibitors, azide and oligomycin. Although 1.5 microM DCCD is ineffective, 150 microM DCCD leads to total inhibition of ATPase activity. The ATPase activity of goblet cell apical membranes is stimulated not only by K+, but also, in order of decreasing effectiveness, by Rb+, Li+, Na+ and even Mg2+. Replacement of Cl- by Br-, F- and HCO3- has less influence than variation of the cations. However, replacement of Cl- by NO3- inhibits strongly this ATPase activity. The ATPase activity described above is characteristic of the alkali metal ion pump containing apical membranes of goblet cells and is not enhanced to a similar degree in other purified midgut epithelial cell plasma membrane segments. Its localization, its broad cation specificity and its insensitivity to ouabain all mimic properties of active ion transport by the lepidopteran midgut and suggest this ATPase as a possible key component of the lepidopteran electrogenic alkali metal ion pump.
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PMID:Cation-stimulated ATPase activity in purified plasma membranes from tobacco hornworm midgut. 293 79


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