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 activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:H+ extrusion by an apical vacuolar-type H(+)-ATPase in rat renal proximal tubules. 131 56

Synaptic vesicles have important roles in the neural transmission at nerve terminals: the storage and the controlled exocytosis of neurotransmitters. At least two different factors are responsible for the concentration process: the vacuolar-type H(+)-ATPase (V-ATPase), establishing an electrochemical gradient of protons, and specific transport systems for transmitters. We will discuss our recent progress on the energy-transducing systems in synaptic vesicles: (1) structural aspects of V-ATPase; (2) energy coupling of transport of transmitters; (3) reconstitution of transporters; (4) effects of neurotoxins and neuron blocking agents; (5) function of synaptic-vesicle-like microvesicles from endocrine tissues.
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PMID:The role of V-ATPase in neuronal and endocrine systems. 136 70

Amoebae of the cellular slime mold Dictyostelium discoideum possess an extensive and dynamic endomembrane system that includes many types of acidic vacuoles. A light membrane fraction from Dictyostelium, rich in vacuolar-type H(+)-ATPase, has been described [Padh, H., Lavasa, M. & Steck, T.L. (1989) J. Cell Biol. 108, 865-874]. Here, we show that this "acidosomal" fraction also contains a high-affinity vanadate-sensitive Ca2+ uptake activity that is stimulated by the pH gradient formed by the H(+)-ATPase. We attribute this Ca2+ uptake to the presence of a H(+)-countertransporting Ca(2+)-ATPase, pumping Ca2+ into an acidic compartment.
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PMID:ATP-driven Ca2+/H+ antiport in acid vesicles from Dictyostelium. 138 10

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.
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PMID:Effect of N,N'-dicyclohexylcarbodiimide on acetylcholine release from Torpedo synaptosomes and proteoliposomes reconstituted with the proteolipid mediatophore. 140 80

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.
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PMID:Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies. 153 25

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.
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PMID:A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane. 183 Dec 2

The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.
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PMID:Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages. 214 29

The role of vacuolar-type H(+)-ATPase (V-ATPase) in the cytotoxic action of diphtheria toxin (DT) was studied by using bafilomycin A1, a specific inhibitor of V-ATPase. Studies with acridine orange showed that the acidification of intracellular acidic compartments was inhibited strongly when Vero cells were treated with 500 nM bafilomycin A1, indicating that bafilomycin effectively inhibits V-ATPase when it is added to the culture medium. The toxicity of DT to Vero cells, which was determined by the inhibition of protein synthesis by DT, was inhibited partially by bafilomycin at 10 nM and inhibited completely at 500 nM. Therefore, V-ATPase is involved in the expression of the toxicity of DT. Studies using 125I-labeled DT showed that bafilomycin inhibited the degradation of internalized DT, indicating that V-ATPase is also involved in this step. Subcellular fractionation revealed that 125I-DT accumulated mainly in the endosome fraction, and not in the lysosome fraction, when the cells were incubated with 125I-DT in the presence of bafilomycin. Under the cell fractionation conditions similar to those used for the DT-treated cells, we determined the location of 125I-labeled epidermal growth factor in the degradation pathway. The result suggests that bafilomycin A1 does not inhibit the transport of epidermal growth factor to lysosome.
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PMID:The cytotoxic action of diphtheria toxin and its degradation in intact Vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase. 214 89

The Saccharomyces cerevisiae gene, VPH1 (Vacuolar pH 1), encodes a 95-kDa integral membrane subunit of the vacuolar-type H(+)-ATPase (V-ATPase) that is required for enzyme assembly; disruption of the VPH1 gene impairs vacuolar acidification (Manolson, M.F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. Chem. 267, 14294-14303). Here we show that STV1 (Similar To VPH1) encodes an integral membrane polypeptide of 102 kDa with 54% identity with the peptide sequence of Vph1p. High copy expression of STV1 partially restores vacuolar acidification in a delta vph1 mutant strain; solubilization and fractionation of membrane proteins from these vacuoles show that Stv1p co-purifies with bafilomycin A1-sensitive ATPase activity and with the 60- and 69-kDa V-ATPase subunits. Immunofluorescence microscopy of strains bearing a single copy of epitope-tagged STV1 reveals punctate staining of the cytoplasm; overexpression of epitope-tagged Stv1p reveals both punctate cytoplasmic staining and vacuolar membrane staining. Northern analysis shows that disruption of STV1 does not affect the level of transcription of VPH1 and that disruption of VPH1 does not affect the level of transcription of STV1. Strains bearing disruption of genes encoding other V-ATPase subunits (VMA1, VMA2, VMA3, and VMA4) fail to grow on media supplemented with 100 mM CaCl2 or 4 mM ZnCl2, media buffered to pH 7.5, or media with a glycerol carbon source. On the same types of media only a delta vph1 delta stv1 double disruption mutant has growth phenotypes equivalent to strains bearing a single disruption of the VMA1, VMA2, VMA3, and VMA4 genes; a delta vph1 strain has only moderate growth inhibition while a delta stv1 strain has wild type growth on the conditions listed above. We conclude that Stv1p is a functional homologue of Vph1p and suggest that Stv1p and Vph1p may be equivalent subunits for V-ATPases located on different organelles. The function of these 100-kDa homologues may be to target or regulate other common V-ATPase subunits for two distinct cellular locations.
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PMID:STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. 751 99

Ovarian oocytes of the prosobranch mollusc Patella vulgata and the pelecypod Ruditapes philippinarum are arrested during prophase of the first maturation division. Release from this blockade, which is revealed by germinal vesicle breakdown, drives these oocytes to a second arrest in metaphase I, at which time the oocytes become fertilizable. The respective roles of Ca2+ and H+ ion movements during this early step in meiosis reinitiation has not been fully established yet. In this work we reveal the presence of acidic vesicles and report that bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, two inhibitors of the vacuolar-type H(+)-ATPase, applied to Ruditapes oocytes, produce a significant inhibition of their response to the natural neurohormone serotonin. Since sodium deprivation did not affect this response, this suggests that a v-type ATPase pump, possibly located in the membrane of these acidic vesicles, may play a subtle role in the cascade of events that releases oocytes from their prophase block. We then describe how 4-aminopyridine, a drug reputed to be a K+ channel antagonist, triggers both meiosis reinitiation and activation of Patella and Ruditapes oocytes. This agent acts as a weak base, its effect depending on external pH. Moreover, using the fluorescent probes BCECF and Fluo-3/AM, we observe that this drug both alkalinizes the endoplasm and promotes an intracellular Ca2+ surge. This dual effect may explain why Ruditapes oocytes no longer stop in metaphase under these conditions and behave like other bivalve species which are directly fertilizable at the germinal vesicle stage.
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PMID:4-aminopyridine acts as a weak base and a Ca2+ mobilizing agent in triggering oocyte meiosis reinitiation and activation in the Japanese clam Ruditapes philippinarum. 757 39


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