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 role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the alpha- and beta-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1, 2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 microM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.
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PMID:Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase. 893 13

We have shown that polarization of an electrogenic H+/K+ ATPase pump located in the secretory (luminal) membrane of the frog gastric mucosa is the major factor contributing to the change in open circuit potential difference (OCPD) induced by voltage clamping. This transmucosal polarization was markedly reduced by H2 blockers famotidine and cimetidine, and by the H+/K+-ATPase inhibitors omeprazole and SCH 28080. SCN-, a nonspecific H+ secretion inhibitor, did not affect the polarization. In the present experiments, the effects of two other inhibitors of H+ secretion were examined, namely, acetazolamide (AA), a carbonic anhydrase inhibitor, and melittin (MEL), an inhibitor of the H+/K+-ATPase enzyme. When AA 10(-3) M or MEL 10(-5) M was added to the nutrient solution, H+ secretion was completely inhibited. While MEL markedly reduced the polarization induced by voltage clamp, AA did not affect the polarization. These data support the concept that MEL directly affects the electrogenic H+/K+-ATPase pump while the inhibition of H+ secretion by AA is by an indirect mechanism. The data further support the electrogenicity of the H+/K+-ATPase.
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PMID:Effect of acetazolamide and melittin on polarization of the frog gastric mucosa proton pump. 898 9

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.
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PMID:Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells. 902 Aug 75

Dopamine-induced natriuretic response which results from the activation of tubular dopamine1 (DA1) receptors is diminished in spontaneously hypertensive rats (SHR). This may be a result of alterations occurring at the receptor level and within the cellular signaling pathway which ultimately causes inhibition of Na+, K(+)-ATPase. There have been reports showing that DA1 receptor induced inhibition of Na+, K(+)-ATPase is abolished in SHR which is due to a decreased activation of PLC and PKC by dopamine. Of the mechanisms, adenylyl cyclase and phospholipase C are two known enzymes linked to DA1 receptors via G proteins. Furthermore, the involvement of phospholipase A2 (PLA2) has also been reported in this process. However, the site of defect in DA1 receptor signaling pathway in SHR is still not well understood. This report will (i) review the coupling of DA1 receptor with G proteins and their levels in Wistar Kyoto (WKY) rats and SHR and (ii) discuss studies dealing with the role of PLA2 in dopamine-induced inhibition of Na+, K(+)-ATPase in WKY rat and SHR kidneys. Fenoldopam, DA1 receptor selective agonist stimulated [35S]GTP gamma S binding in a concentration (10(-9)-10(-4) M)-dependent manner in WKY rats which was attenuated in SHR. Fenoldopam (10 microM)-induced stimulation of [35S]GTP gamma S binding was significantly reduced by a DA1 receptor selective antagonist, SCH 23390 suggesting the involvement of DA1 receptor. Furthermore, the specific antipeptides Gs alpha, and Gq/11 alpha significantly blocked fenoldopam-stimulation of [35S]GTP gamma S binding suggesting the coupling of DA1 receptor with both the G proteins. Western analysis revealed a significant decrease in Gq/11 alpha but no changes in Gs alpha in SHR compared to WKY rats. Dopamine inhibited Na+, K(+)-ATPase activity in a concentration (10(-9)-10(-5) M)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10 microM)-induced inhibition in Na+, K(+)-ATPase activity was significantly blocked by mepacrine (a PLA2 inhibitor) suggesting the involvement of PLA2 in dopamine-mediated inhibition of Na+, K(+)-ATPase. Arachidonic acid (AA), a PLA2 product, inhibited Na+, K(+)-ATPase in a concentration (1-100 microM)-dependent manner in WKY rats while the inhibition in SHR was significantly attenuated (IC50: 7.5 microM in WKY and 80 microM in SHR). Furthermore, lower concentration (1 microM) of AA stimulated the enzyme activity in SHR. This suggests a defect in the metabolism of AA in SHR. Proadifen (10 microM), an inhibitor of cytochrome P-450 monoxygenase (an arachidonic acid metabolizing enzyme) significantly blocked the inhibition produced by arachidonic acid in WKY rats and abolished the difference in arachidonic acid inhibition of Na+, K(+)-ATPase between WKY rats and SHR. These data suggest that (i) the reduced activation of G proteins following DA1 receptor stimulation, (ii) reduced amount of Gq/11 alpha and (iii) a defect in the AA metabolism may be responsible for the reduced dopaminergic inhibition of sodium pump activity and a diminished natriuretic response to dopamine in SHR.
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PMID:Dopamine-1 receptor G-protein coupling and the involvement of phospholipase A2 in dopamine-1 receptor mediated cellular signaling mechanisms in the proximal tubules of SHR. 902 41

The distal colon absorbs K+ (JK) and secretes H+ (JH) by what is thought to be an H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). However, the colonic ATPase differs structurally and functionally from the gastric H(+)-K(+)-ATPase. To evaluate the link between JH and JK, JH and JK were simultaneously measured with ion-specific electrodes in segments of rat distal colon. JH and JK averaged 0.40 +/- 0.03 and 0.30 +/- 0.03 mu eq.h-1.cm-2 (n = 191), but JH and JK did not correlate (r = 0.005, not significant). The gastric H(+)-K+ pump inhibitors SCH-28080 (100 microM) and omeprazole (100 microM), as well as a vacuolar H(+)-ATPase inhibitor, bafilomycin A1 (10 microM), did not affect JH or JK. However, the Na(+)-K(+)-ATPase inhibitors ouabain (1 mM) and N-ethylmaleimide (10 microM) inhibited JK but not JH. Although 1 mM orthovanadate inhibited both JH and JK, at lower concentrations orthovanadate only affected JK. Furthermore, removing K+ from the medium did not affect JH. Secondary hyperaldosteronism increased both JH and JK; however, ouabain (1 mM) reduced JK but not JH. Cl(-)-free medium inhibited voltage-insensitive JH and voltage-sensitive JK. Medium pH affected JH, but that effect was contrary to the effect that pH had on Rb+ flux. These data failed to identify a relationship between JH and JK and appear to suggest that JH and JK occur by separate pathways.
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PMID:Net H+ and K+ fluxes across the apical surface of rat distal colon. 903 76

This study sought to investigate the presence and characteristics of K+-ATPase activity in chicken intestinal epithelia. A cytochemical method revealed Na+-independent, ouabain-sensitive, K+-ATPase activity in the apical, but not in the basolateral, membrane of chicken colonic and caecal epithelial cells. K+-ATPase activity was not observed in the small intestine. The measurement of K+-activated pNPPase activity was used to characterize the K+-ATPase activity evidenced by the cytochemical method. In addition, K+ and NH4+, but neither Na+ nor Li+, could activate pNPPase activity in chicken intestinal epithelia. Vanadate abolished ouabain-sensitive, K+-activated pNPPase activity in the three membrane preparations tested, whereas oligomycin and SCH 28080 were without effect. The Km for K+ and the ouabain IC50 values for the apical colonic and caecal K+-activated pNPPase activity were higher than those measured for K+-activated pNPPase activity measured in the basolateral membrane of chicken jejunal enterocytes. The results indicate that the apical membranes of chicken colon and caecum possess Na+-independent, ouabain-sensitive K+-activated-ATPase activity.
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PMID:Apical ouabain-sensitive K+-activated-ATPase activity in colon and caecum of the chick. 906 49

We found that isolated gastric vesicles contain a novel Mg2+-ATP-dependent phospholipid translocation (flippase) activity. Fluorescence analogue of phosphatidylcholine, 2-(12-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3- phosphocholine, was ATP-dependently translocated from the outer (cytosolic) to inner (luminal) leaflet of the lipid membrane bilayer of hog gastric vesicles. The translocation was saturable and depended on time and the ATP concentration (Km = 3.1 microM). The basal Mg2+-ATPase activity of gastric vesicles in the absence of K+ showed high (Km = 1.6 microM) and low (Km = 80 microM) affinities for ATP, indicating that the present flippase activity is driven mostly by the high affinity Mg2+-ATPase activity. It required Mg2+ but not K+. Verapamil, which is an inhibitor of mouse mdr2 phosphatidylcholine flippase, did not inhibit the present flippase activity. Isolated sarcoplasmic reticulum vesicles that contain Ca2+-ATPase did not show any flippase activity. Fluorescence analogues of phosphatidylserine and phosphatidylethanolamine were similarly translocated by the gastric flippase. These phospholipid flippase activities were inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) (IC50 = 0.14-0.25 microM), a specific K+-ATPase inhibitor of gastric H+,K+-ATPase rich in gastric vesicles. IC50 value for the SCH 28080-inhibitable Mg2+-ATPase activity was about 0.13 microM, indicating that the phospholipid translocation was driven mostly by the SCH 28080-sensitive Mg2+-ATPase activity. Possible physiological roles of flippases were discussed in relation with the gastric acid secretory and cytoprotective mechanisms.
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PMID:The phospholipid flippase activity of gastric vesicles. 909 84

A compound, SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo [1,2-a]pyridine-3-acetonitrile), reversibly inhibits gastric and renal ouabain-insensitive H+,K+-ATPase, but not colonic ouabain-sensitive H+,K+-ATPase. By using the functional expression system and site-directed mutagenesis, we analyzed the putative binding sites of SCH 28080 in gastric H+,K+-ATPase alpha-subunit. It was previously reported that the binding site of SCH 28080, which is a K+-site inhibitor specific for gastric H+,K+-ATPase, was in the first extracellular loop between the first and second transmembrane segments of the alpha-subunit; Phe-126 and Asp-138 were putative binding sites. However, we found that all the mutants in the first extracellular loop including Phe-126 and Asp-138 retained H+, K+-ATPase activity and sensitivity to SCH 28080. Therefore, amino acid residues in the first extracellular loop are not directly involved in the SCH 28080 binding nor indispensable for the H+, K+-ATPase activity. Here we propose a candidate residue that is important for the binding with SCH 28080, Glu-822 in the sixth transmembrane segment. Mutations of Glu-822 to Asp and Ala (mutants termed E822D and E822A, respectively) decreased the ATPase activity to about 45% and 35% of the wild-type enzyme, respectively, while the mutations to Gln and Leu abolished the activity. Mutant E822A showed a significantly lower affinity for K+ than the wild-type enzyme, indicating that Glu-822 is involved in determining the affinity for K+. The sensitivity of mutant E822D to SCH 28080 was 8 times lower than that of the wild-type enzyme. The counterpart of Glu-822 in gastric H+,K+-ATPase is Asp in Na+,K+-ATPase and other colonic ouabain-sensitive H+,K+-ATPase, which are insensitive to SCH 28080. These results suggest that Glu-822 is one of important sites that bind with SCH 28080.
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PMID:Mutational analysis of putative SCH 28080 binding sites of the gastric H+,K+-ATPase. 921 17

The ouabain-sensitive, K-stimulated p-nitrophenyl phosphatase (K-pNPPase) activity, an associated activity of the Na,K-ATPase, was assayed in tentacles of the sea anemone Stichodactyla helianthus to investigate the possibility that the sea anemone Na,K-ATPase activity is an associated activity of an H,K-ATPase. Activity was maximal at pH 6.5-7.0, decreasing only slightly in acidic medium but falling abruptly in alkaline medium to 60% of maximum at pH 7.4. The pH of maximum activity was not remarkably altered in high ionic strength medium (560 mM choline chloride), but ouabain-sensitive K-pNPPase activity of both rat and sea anemone was strongly inhibited. Inhibitors of the gastric H,K-ATPase, 100 microM omeprazole and 10 microM SCH 28080, did not inhibit the ouabain-sensitive K-pNPPase activity. Activity of the sea anemone enzyme was inhibited by 10 microM ammonium vanadate, an inhibitor of P-type ATPase, and not by 2.5 mM sodium azide, an inhibitor of both F-type and V-type ATPase. Because the sea anemone K-pNPPase activity was previously found to be more sensitive to ouabain than the Na,K-ATPase activity, K(+)-ouabain antagonism was investigated and found to be relatively muted, whereas K(+)-Na+ competition was stronger than in the rat kidney.
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PMID:Effect of high ionic strength and inhibitors of H,K-ATPase on the ouabain-sensitive K-p-nitrophenylphosphatase activity in the sea anemone Stichodactyla helianthus. 922 81

The enzyme catechol-O-methyltransferase (COMT), which plays an important role for dopamine metabolism, is abundantly expressed in the kidney. To test whether the natriuretic effects of dopamine may be related to the rate of dopamine metabolism, rats were treated with nitecapone, a peripheral inhibitor of COMT. Nitecapone, given by gavage, induced a highly significant (5.6-fold) increase in sodium excretion, which was associated with an inhibition of the Na+,K+-ATPase activity in both the proximal convoluted and proximal straight tubules (PCT and PST, respectively). These effects were completely abolished if the rats were also treated with a specific dopamine 1 antagonist, SCH 23390. Furthermore, the natriuretic effect of nitecapone was also observed in rats on a high salt diet. The kidney-specific pro-drug to dopamine, glu-dopa, induced a significant, but less pronounced increase in urinary sodium excretion, associated with a dopamine-dependent inhibition of the Na+,K+-ATPase activity in the PCT but not in the PST. Nitecapone and glu-dopa had an additive natriuretic effect. It is concluded that COMT plays an important role in determining the natriuretic effects of the renal dopamine system.
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PMID:Inhibition of COMT induces dopamine-dependent natriuresis and inhibition of proximal tubular Na+,K+-ATPase. 929 Nov 95


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