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 synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.
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PMID:Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle. 132 24

We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).
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PMID:A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat. 133 44

Mechanisms of gastric parietal cell secretory membrane Cl- transport and the role of this Cl- transport in acid secretion were investigated by examining the effects of two Cl- channel blockers, diphenylamine-2-carboxylate (DPC) and 9-anthracene carboxylate (9-AC) on acid secretion using isolated, enriched rabbit parietal cells. Resting and stimulated acid secretion in intact cells (measured as [14C]aminopyrine accumulation) was inhibited by DPC and 9-AC, irrespective of agonist used. Apparent inhibition constants (Ki) were 2.4 x 10(-4) M for DPC and 1.2 x 10(-3) M for 9-AC for all responses. Digitonin-permeabilized parietal cells were used to bypass possible inhibitory effects of these compounds on basolateral membrane transport processes and to investigate effects only on the secretory membrane. Both blockers inhibited ATP-driven acid secretion in resting and stimulated permeable cells with apparent Ki values in the same range as measured in intact cells, suggesting that the site of action of these blockers is at the secretory membrane. H(+)-K(+)-ATPase activity in situ in permeable parietal cells, measured as 2-methyl-8-(phenylmethoxy)imidazo(1,2) pyridine-3-acetonitrile (SCH28080)-inhibitable ATP hydrolysis, was higher in stimulated compared with resting cells. Addition of 10 mM NH4Cl abolished this difference, and maximal H(+)-K(+)-ATPase activity was measured. SCH28080 and NH4Cl each abolished both resting and stimulated acid accumulation. DPC and 9-AC inhibited resting and stimulated H(+)-K(+)-ATPase activities, without exerting inhibitory effects on the enzyme itself, since the blockers had no effect on maximal NH4(+)-stimulated H(+)-K(+)-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cl- channel blockers inhibit acid secretion in rabbit parietal cells. 169 27

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.
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PMID:Phosphorylation of H+/K(+)-ATPase by inorganic phosphate. The role of K+ and SCH 28080. 184 26

Na+, K(+)-ATPase inhibitory activity in urine fractionated by HPLC was quantified in 7 normotensive male subjects during changes in dietary sodium intake. Subjects were studied on free sodium intake for 2 days, on low sodium intake (2 g/day) for 3 days, on high sodium intake (22 g/day) for 4 days and subsequently on normal sodium intake (6 g/day) for 2 days. Na+, K(+)-ATPase inhibitory activity in fraction 10 eluted with 17% acetonitrile by reverse-phase HPLC was 12.3 +/- 5.2% (mean +/- S.D.) on free sodium intake, 8.7 +/- 9.8% on the 3rd day of low sodium intake, 61.2 +/- 6.6% on the 4th day of high sodium intake, and 20.5% +/- 0.7% on the 2nd day of the normal sodium intake. Changes in Na+, K(+)-ATPase inhibitory activity of fraction 10 were closely associated with those in urinary sodium excretion. These results suggest that an endogenous Na+, K(+)-ATPase inhibitor(s) which plays a physiological role in the control of sodium and water balance may exist in this particular fraction.
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PMID:Na+, K(+)-ATPase inhibitory activity of fractionated urine during changes in dietary sodium intake in man. 214 68

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.
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PMID:Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase. 216 16

A fully automated high-performance liquid chromatographic method is described for the determination of the new H+/K+ ATPase inhibitor BY 1023/SK&F 96,022 and its major metabolite occurring in dog serum. The method uses direct sample injection of up to 200 microliters and a pre-column switching technique. In order to optimize the recovery, pre-column conditions were varied systematically with respect to the pH of the pre-column eluent, its buffering capacity and content of acetonitrile. Optimization resulted in near 100% recovery for both compounds, thus allowing the use of external standardization. The linearity range, precision and detection limits were determined and the method shown to be applicable to both serum and plasma. The method was applied to define the pharmacokinetics in dogs and humans.
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PMID:High-performance liquid chromatographic determination of the H+/K+ ATPase inhibitor (BY 1023/SK&F 96,022) and its sulphone metabolite in serum or plasma by direct injection and fully automated pre-column sample clean-up. 217 66

Hog gastric vesicles showed Cl- conductance when treated with Cu2+-o-phenanthroline, an S-S cross-linking reagent. An IgG monoclonal antibody caused dose-dependent inhibition of Cl- conductance that had been induced by S-S cross-linking. The antibody did not cause intervesicular aggregation, as determined by measurement of vesicle size. These results show that Cl- conductance, the stimulation and inhibition of which are regulated reversibly by S-S----2SH transformation, is due to native, physiological channels. The antibody also dose dependently inhibited the activities of H,K-ATPase and p-nitrophenyl phosphatase in gastric vesicles, but did not inhibit Na,K-ATPase obtained from dog kidney. Immunoblotting with the antibody of vesicle proteins solubilized in sodium dodecyl sulfate-polyacrylamide gel showed that the antibody binds to a 95-kDa subunit of H,K-ATPase and its dimeric 180-kDa polypeptide. The antibody-binding sites of H,K-ATPase activity and the Cl- channel for the inhibition were present on the external (cytosolic) surface of the transmembraneous ATPase. A gastric antisecretory compound, 2-methyl-8-(phenylmethoxy)imidazo[1,2 alpha] pyridine-3-acetonitrile (SCH 28080), competitively bound to the high affinity site of K+ on the internal (luminal) surface of H,K-ATPase, and its half-maximal inhibitory concentration for H,K-ATPase activity in tight vesicles was 0.2 microM in the presence of valinomycin. SCH 28080 also dose dependently inhibited opening of Cl- channels by S-S cross-linking, the regulatory site being present on the cytosolic side and more internally than the antibody binding site. The half-inhibitory concentration of SCH 28080 was 0.3 microM. The present results with the antibody and SCH 28080 indicate that the Cl- channel is part of the function of H,K-ATPase.
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PMID:The Cl- channel in hog gastric vesicles is part of the function of H,K-ATPase. 244 90

2-Methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH 28080) is a freely reversible K+ site inhibitor of the gastric (H+ + K+)-ATPase. In the presence of 2 mMMgSO4, [14C]SCH 28080 bound saturably to gastric vesicle preparations containing the (H+ + K+)-ATPase and was displaced by lumenal K+. A binding stoichiometry of 2.2 +/- 0.1 mol of SCH 28080/mol of catalytic phosphorylation sites was observed. The affinity of SCH 28080 binding was increased approximately 10-fold (to 45 nM) in the presence of 2 mM ATP. High affinity binding also occurred with 2 microM ATP but not with up to 200 microM D-[beta, gamma-CH2]ATP, suggesting that high affinity binding was to a phosphorylated form of the enzyme. In the presence of ATP, the association rate constant was linearly related to the concentration of SCH 28080. However, the association and dissociation rates of SCH 28080 binding were slow, especially at low temperature (at 1.5 degrees C half-maximal binding of 50 nM SCH 28080 was calculated to occur after 232 s). Binding appeared to be predominantly entropy driven with a high activation energy (40 kJ/mol at 37 degrees C). In the absence of ATP, the association rate constant was not linearly related to the concentration of SCH 28080, suggesting that a conformational change in the enzyme was required before binding could occur.
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PMID:The binding of a K+ competitive ligand, 2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile, to the gastric (H+ + K+)-ATPase. 253 22

Definition of the interrelationship between the conformational characteristics of a series of substituted imidazo[1,2-a]pyridines and their antiulcer activity was investigated by examining the conformational properties of 3-cyano-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine (1), using a variety of experimental and theoretical methods. The results of these studies was the identification of two distinctly different candidates, designated the "folded" and the "extended" conformation, respectively, to represent the two possible minimum-energy conformations of 1. In order to select the biologically relevant conformer, a group of 3-substituted 2-methylimidazo[1,2-a]pyridines, having either a cis or a trans 2-phenylethenyl substituent at the 8-position were designed as conceptually simple and synthetically accessible semirigid analogues of the respective candidate conformers. Gastric antisecretory activity was found to reside only in the trans isomers (compounds 11, 15, and 17), which mimic the "extended" conformation. This observation led to the construction of 8,9-dihydro-2-methyl-9-phenyl-7H-imidazo[1,2-a]pyrano[2,3-c]pyridi ne-3- acetonitrile (40), a rigid tricyclic analogue that is effectively locked in the "extended" conformation and that exhibited an antiulcer profile comparable to that of prototype 1. These results unequivocally demonstrate that, in accord with expectation for a drug operating at a specific receptor, the conformational characteristics of the molecule have a substantial effect in determining its antiulcer activity. More precisely, it has been demonstrated that it is the "extended" conformation of 1 that represents the "bioactive" form of the drug. These results constitute the basis for a molecular probe that should aid in the investigation of the as yet uncharacterized gastric proton pump enzyme (H+/K+-ATPase), by means of which 1 and its analogues presumably exert their pharmacologic actions.
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PMID:Antiulcer agents. 4. Conformational considerations and the antiulcer activity of substituted imidazo[1,2-a]pyridines and related analogues. 275 93


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