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 degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 125I-insulin in isotonic KCl at 30 degrees C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5-6 (t1/2: 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglycol). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5-8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (delta pH about 0.8-0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of Cl-, other anions being less effective (Br- greater than gluconate = SO4(2-) greater than NO3- = sucrose = mannitol) and/or inhibitory (NO3-). Na+, K+ and Li+ supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2+ = Mn2+ greater than Co2+ greater than Ni2+ = Zn2 greater than Ca2+). Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5-9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulin-receptor complex, is required for optimum degradation of insulin within liver endosomes.
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PMID:Degradation of insulin in isolated liver endosomes is functionally linked to ATP-dependent endosomal acidification. 214 19

The Ca2(+)-dependent adenosinetriphosphatase (Ca2(+)-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is phosphorylated by inorganic phosphate (Pi) in the absence of Ca2+. The reaction can be described by the following simplified scheme: [formula: see text] where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E.Pi is a noncovalent and acid-labile complex. The reaction is Mg2(+)-dependent. Membrane fragments deposited on Millipore filters were successively perfused with two solutions, at constant flow. The effluent samples were analyzed. The perfused solutions were Ca2+ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [32P]Pi, 32P binding is only detected in the presence of Mg2+, indicating the formation of the phosphoenzymes (E.Pi and E-P). Following perfusions of the phosphoenzymes with 5% trichloroacetic acid, 32P release indicates the amount of the acid-labile moiety (E.Pi). After phosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 +/- 0.12, and 1.48 +/- 0.10 nmol of Pi/mg of protein ( +/- S.E., n = 5), after phosphorylations with 20 microM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2(+)-ATPase cycle. Keq = k2/k-2), in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg.Pi complex, since Mg2+ is necessary for step 1 in the above scheme.
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PMID:Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2(+)-dependent adenosinetriphosphatase. 214 71

By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.
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PMID:Chinese hamster ovary cell mutants defective in myo-inositol transport. 221 83

The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, M*ATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or M*ATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.
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PMID:Reaction intermediates formed by myofibrils during the ATPase reaction under relaxed conditions. 252 74

Ribonucleic acid was isolated from the fundic gastric mucosae of rats and rabbits by cesium chloride centrifugation of guanidine isothiocyanate-denatured mucosal homogenates, and poly A+ RNA was recovered from the pellets by oligodeoxythymidine column selection. When added to rabbit reticulocyte lysates, this poly A+ RNA stimulated [35S]methionine incorporation into trichloroacetic acid-precipitable material. Fluorographic analysis of the lysates showed protein synthesis to be dominated by polypeptides with molecular weights from 40,000 to 50,000, presumably prepepsinogen isoforms. Immune precipitation of the lysates with monoclonal antibodies directed against the gastric H+,K+-adenosine triphosphatase yielded bands at 94 kilodaltons and more diffuse banding at 180 kilodaltons. Further purification of the poly A+ RNA on sucrose gradients eliminated prepepsinogen messenger RNA; nascent H+,K+-adenosine triphosphatase synthesized by purified messenger RNA consisted of polypeptides with molecular weights between 88,000 and 94,000. The study indicates that cell-free translation of gastric mucosal messenger RNA may provide a useful model for analysis of gastric H+,K+-adenosine triphosphatase biosynthesis and processing.
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PMID:Cell-free synthesis of rat and rabbit gastric proton pump. 255 Mar 9

The rapid release of 86Rb from an occluded state of the Na,K-ATPase was studied in a rapid filtration apparatus. In the presence of Mg2+, 86Rb release was found to be stimulated by arsenate and thiophosphate just as it is stimulated by Pi. The affinity of Na,K-ATPase for arsenate is about 4-fold higher than that for Pi and the affinity for thiophosphate is about 5-fold lower than that for Pi. With all three divalent anions, the rates of maximally stimulated 86Rb release were constant between pH 6.5 and 7.5, and decreased between pH 7.5 and 8.5. The affinity for phosphate and thiophosphate increased in the latter pH range, while the affinity for arsenate decreased. The results are not consistent with titration of the divalent anion as the sole determinant of effectiveness in stimulating 86Rb release; thus they suggest that titration of groups on the protein is important. A delay in the rise to the maximum rate of 86Rb release upon stimulation with arsenate is shown to be due to the time required for arsenylation, and from an analysis of the rise and fall of the rate of 86Rb release the rate constants for arsenylation and dearsenylation at pH 7.2 can be estimated; the decay in the rate of 86Rb release when arsenate or phosphate is removed from the solution provides a second method for determination of the dearsenylation rate. The dearsenylation rate constant increases 5-fold from pH 6.1 to 7.5. From the time course of 86Rb release in the presence of Pi we estimate that the rate of dephosphorylation is 50-100 s-1 at pH 6.6 and 20 degrees C; at pH 7-7.5 the rate is too fast to determine. Dimethyl sulfoxide (25%) increases the affinity for arsenate (or phosphate), due to reciprocal changes in arsenylation and dearsenylation rates, and it increases the rate of 86Rb release 2-3 fold. Finally, the level of phosphointermediate formation from 32Pi was determined in the absence and presence of K+: when methanol is used as a denaturant, K+ has only a small effect on the observed level of E-32P, but when trichloroacetic acid is used, K+ is found to reduce the observed level to less than 50% of the control value.
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PMID:Rapid 86Rb release from an occluded state of the Na,K-pump reflects the rate of dephosphorylation or dearsenylation. 283 3

Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low temperature. A phosphorylated compound was rapidly formed which was stable in 10% trichloroacetic acid at 0 degree C. The lability of this compound in the presence of hydroxylamine or hot trichloroacetic acid indicated an acylphosphate, i.e., an ATPase phosphointermediate. Vanadate but not N-ethylmaleimide inhibited the formation of this derivative. Since the ATP-dependent generation of a transmembrane potential in chromaffin granule vesicles by the H+-pump was inhibited by N-ethylmaleimide but not by vanadate, the acylphosphate should not be associated with the H+-pump, i.e. ATPase I. We suggest that it is associated with ATPase II, an ATPase of unknown function present in chromaffin granule membrane preparations. This hypothesis is supported by the fact that ATPase II is vanadate sensitive and has a molecular mass of 140 kDa, properties similar to those of the phosphorylated intermediate.
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PMID:The acylphosphate present in chromaffin granule membrane preparations is not associated with the proton-pump. 287 89

A fluorometric micro protein assay based on fluorescamine-labelling of homogeneous proteins in solution has been developed which is capable of accurately quantitating as little as 25 ng protein at a concentration of 1.25 micrograms/ml. This micro assay uses a flow-through HPLC fluorescence detector. Typical micro assays measuring bovine serum albumin standards (0-25 mg/l) yielded linear regression coefficients of r = 0.999. Assays of purified Ca2+-ATPase solutions determined by the micro fluorescamine procedure correlated well with measurements made using the deoxycholate-TCA-precipitation modification of the Lowry assay: 1.0 microgram ATPase by Lowry method = 1.1 microgram protein by fluorescamine microassay (when both procedures were standardized with bovine serum albumin) (r = 0.995). The assay proposed offers a 100-fold increase in sensitivity, compared to the Lowry procedure.
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PMID:Fluorometric determination of nanogram quantities of protein in small samples: application to calcium-transport adenosine triphosphatase. 294 Nov 85

1. Microsomes prepared from guinea-pig and ox brain were incubated for periods of a few seconds with low concentrations of Mg-[(32)P]ATP, the reaction was stopped with trichloroacetic acid and determinations were made of the phosphate bound to the acid-washed, and in some cases solvent-extracted, residue. 2. At 20 mum-ATP, at 37 degrees and in the presence of Na(+) ions, 30-50 mumumoles of phosphate/mg. of microsomal protein were bound by the preparation within 1 sec. of starting the reaction; little further change in level occurred until hydrolysis of ATP exceeded 50%, when the bound phosphate began to decline fairly rapidly to the zero-time value. 3. At 20mum-ATP without Na(+) ions present or in the presence of K(+) ions, the level of bound phosphate increased gradually and did not decline as ATP hydrolysis approached completion. 4. Potassium ions either inhibited the formation of Na(+)-dependent bound phosphate or, when added during the course of the reaction, rapidly reduced its level. 5. At 200 mum-ATP the bound phosphate formed in the presence of Na(+) ions appeared to consist of a mixture of the unstable Na(+)-dependent type and the stable type requiring only Mg(2+) ions for its formation. 6. Non-radioactive ATP added during the course of the reaction at 20 mum-ATP with Na(+)ions present rapidly discharged virtually all the bound (32)P counts; at 200 mum-ATP only a proportion of the label was similarly discharged. The Na(+)-dependent bound phosphate is therefore turning over, in contrast with that formed in the absence of Na(+)ions, which proved more stable. 7. The Na(+)-dependent bound phosphate was not in the form of ATP; experiments with [(14)C]ATP instead of [(32)P]ATP showed a small and invariable binding of ATP by the preparation unaffected by Na(+) ions or time of incubation. 8. Under the usual conditions employed in this work ouabain stimulated formation of Na(+)-dependent bound phosphate when Na(+) ions were suboptimum and inhibited it when optimum Na(+) ions were present. 9. The Na(+)-dependent binding reaction under present conditions did not involve incorporation into phosphorylserine groups. 10. The relation of the findings to the (Na(+),K(+))-ATPase of the preparation, and to observations in brain slices appearing to implicate phosphorylserine groups in cation transport, is discussed.
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PMID:Phosphate binding by cerebral microsomes in relation to adenosine-triphosphatase activity. 422 16

1. Ox-brain microsomes were incubated with [gamma-(32)P]ATP under various conditions. After the reaction, which was stopped with trichloroacetic acid, a small amount of phosphate remained bound to the washed precipitate. 2. Properties of the bound phosphate were studied by treatment with buffers and solvents. 3. The Na(+)-dependent increment in bound phosphate, predominant at low ATP concentration and features of which suggest involvement in the concomitant adenosine-triphosphatase activity, was rapidly released in both circumstances. 4. In aqueous media the labile phosphate was released entirely as inorganic phosphate at faster rates with increasing alkalinity. 5. In acidified chloroform-alcohol mixtures the released phosphate appeared both as inorganic phosphate and different single (32)P-labelled organic phosphates, which were tentatively identified as the relevant mono-alkyl phosphates, presumably derived by acid-catalysed alcoholysis of a labelled microsomal component, or components. 6. The labile phosphate corresponded to the P exchangeable with non-radioactive ATP added during the enzyme reaction. 7. The possible molecular nature of the labile fraction of the bound phosphate is discussed.
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PMID:Properties of phosphate bound to cerebral microsomes during adenosine-triphosphatase activity. 422 17


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