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)

A maize-root microsomal fraction was enriched in ATPase by treatment with Triton X-100. This activity, which reached 1.2-2.0/mumol Pi x min-1 x mg protein-1, was specific for ATP, very slightly stimulated by K+, inhibited by orthovanadate and diethylstilbestrol, resistant to oligomycin and azide, and had a Km of 1.2 mM MgATP. Incubation of the microsomal fraction with [gamma 32-P]ATP followed by electrophoresis in acid conditions revealed the presence of several phosphoproteins. The phosphorylation of a 110000-Mr polypeptide reached the steady-state level in less than 5 s and rapidly turned over the phosphate group. The phosphorylation level was an hyperbolic function of the [ATP] with a Km of 0.6 mM, suggesting that the rate of Pi production was proportional to the phosphoprotein concentration. The extent of phosphoprotein was decreased by vanadate and diethylstilbestrol. The phosphorylation level was 30% decreased by 50 mM K+ or Na+ while the ATPase activity was slightly stimulated (12% and 5%, respectively). The polypeptide could not be phosphorylated in reverse by Pi. This phosphorylated intermediate from maize-root microsomes exhibits molecular properties characteristic of transport ATPases such as the yeast plasma membrane H+-translocating ATPase. This similarity indicates existence of a transport ATPase in plant plasma membranes. Three other plant microsomal polypeptides (Mr = 52000, 17000 and 16000) and a low molecular weight component (Mr less than 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the Mr less than 1000 phosphorylated component were inhibited by vanadate and diethylstilbestrol. These properties are similar to those of the protein kinase activity recently described in yeast plasma membranes.
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PMID:Phosphorylated intermediate of a transport ATPase and activity of protein kinase in membranes from corn roots. 622 27

A partially purified preparation of the plant plasma membrane ATPase was phosphorylated when incubated with [gamma-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. The sensitivity of the phosphoenzyme bond to alkaline pH and to hydroxylamine indicates that it is an acylphosphate. Both the ATPase activity and the phosphorylation of the enzyme exhibited an apparent Km value of 0.3 mM ATP. When the phosphorylated enzyme was analyzed by electrophoresis in sodium dodecyl sulfate, only one major band with a molecular weight of about 105,000 contained radioactivity. These results indicate that the plant plasma membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal and fungal plasma membranes.
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PMID:Phosphorylated intermediate of the ATPase of plant plasma membranes. 622 52

In the presence of Mg2+ or Ca2+ the membranes of the anaerobic glycolytic bacterium Lactobacillus casei hydrolyze 0.1-0.2 mumole ATP/min/mg of protein with a pH optimum 6.4. This activity is inhibited by N,N'-dicyclohexylcarbodiimide and is insensitive to oligomycin, ouabain, vanadate and hydroxylamine. A soluble ATPase was isolated and purified from L. casei membranes. The specific activity of this ATPase is 3.0-4.0 mumole ATP/min/mg of protein. The enzyme homogeneity was established by analytical polyacrylamide gel disc electrophoresis and by analytical centrifugation (S20, omega = 12 +/- 0,5). The molecular weight of the enzyme is 270 000. Polyacrylamide gel electrophoresis of ATPase denaturated by 1% SDS and 8 M urea in the presence of SDS revealed one type of subunits with Mr = 43 000. These subunits could not be separated by isoelectrofocusing in polyacrylamide gel in the presence of 8 M urea and migrated as a single peptide with pI at 4.2. The experimental results suggest that the soluble ATPase from L. casei consists of six identical subunits with Mr of 43 000.
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PMID:[Characteristics of subunit composition of H+-ATPase from the anaerobic bacterium Lactobacillus casei]. 622 21

The calcium-dependent acylphosphate formed by the calcium transport ATPase of cardiac sarcoplasmic reticulum and the calcium-, calmodulin-dependent phosphoester(s) of sarcoplasmic reticulum fractions formed by a calcium-, calmodulin-dependent membrane-bound protein kinase can be distinguished by removal of calcium and/or magnesium by EDTA or hydroxylamine treatment of the acid denaturated membranes. Both procedures decompose the acylphosphate with little effect on the phosphoester(s). Calmodulin-dependent phosphorylation (2.44 nmol/mg SR protein) reduces the apparent K(Ca) of the acylphosphate steady state level of the calcium transport ATPase from 0.56 to 0.34 microM free calcium, without affecting the maximum phosphoenzyme level (0.93 versus 0.89 nmol/mg protein), and has little, if any, effect on the Hill-coefficient (1.32 versus 1.54).
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PMID:Alteration of acylphosphate formation of cardiac sarcoplasmic reticulum ATPase by calmodulin-dependent phosphorylation. 623 2

The complete amino acid sequence of dicyclohexylcarbodiimide (DCC)-binding subunit of proton adenosine triphosphatase from glycolysing bacteria Streptococcus faecalis was established. Isolation of the protein from subbacterial particles was carried out by using extraction with a chloroform/methanol mixture and following gel-filtration on Sephadex LH-60 and Bio-gel P-30. To establish the primary structure, use was made of cyanogen bromide and hydroxylamine cleavages, trypsin and partial acid hydrolyses. Separation of the peptide fragments obtained from cyanogen bromide and hydroxylamine cleavages and partial acid hydrolysis was performed by gel-filtration on Bio-gel P-10 and reversed-phase HPLC. Peptide structures were determined mainly with the aid of 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate. The polypeptide chain of the protein consists of 71 amino acid residues (mol. wt. 7291). The primary structure of the protein from S. faecalis shares all common features of the structural organization of other H+-ATPase DCC-binding subunits and shows a high degree of homology with the corresponding subunit of thermophilic bacterium PS-3. Inactivation of H+-ATPase with DCC was due to modification of Glu54 of the polypeptide chain.
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PMID:[Primary structure of the dicyclohexylcarbodiimide-binding subunit of Streptococcus faecalis H+-ATPase]. 623 59

Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant, Km (1.5 x 10(5) M), similar to the inhibitory constant KI (3 X 10(-5) M), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabaiation rate constants reported for other tissues and species. The high dissociation rate constant 3.6 x 10(-2) sec-1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represents a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.
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PMID:Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes. 624 95

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.
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PMID:Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates. 625 Aug 46

The effects of phenylalanine (PHE) and its deaminated metabolites phenylpyruvate (PHP), phenyllactate (PHL) and phenylacetate (PHA) on sodium and potassium activated adenosinetriphosphatase (Na+,K+-ATPase) in synaptosomes from rat brain were investigated. At very low concentrations (5-10 microM). PHE, PHL and PHA inhibited the activity, while PHP stimulated the activity. At intermediate concentrations (50-100 microM), all compounds had no effect, but at higher (0.5-1.0 mM) concentrations they inhibited the enzyme activity. Thus all the compounds tested showed a biphasic effect on the enzyme activity. Hydroxylamine inhibited the Na+,K+-ATPase activity when present alone; simultaneous addition of hydroxylamine and PHE, however, eliminated the inhibitory effects of each other. Reversal of mutual inhibition also occurred in the presence of hydroxylamine and very low (5-10 microM) concentrations of PHL or PHA. The inhibitory effects of PHE at aLl concentrations, and of PHL or PHA at low concentrations, were also eliminated in the presence of EGTA. The data indicate that inhibition of brain membrane Na+,K+-ATPase by PHE and by low concentrations of PHL and PHA may involve metal ions, but that the inhibition by high concentrations of these metabolites must occur by a different mechanism. Since Na+,K+-ATPase plays a central role in neuronal function, and the presence of excess PHE and its deaminated metabolites occurs in brain tissue under conditions of experimentally induced hyperphenylalaninemia and genetic phenylketonuria, the neurologic impairment in experimental and genetic PKU may in part be related to the deleterious effects of these compounds on brain ATPase.
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PMID:Effects of phenylalanine and its deaminated metabolites on Na+,K+-ATPase activity in synaptosomes from rat brain. 628 50

1) Treatment of (Na+ + K+)-ATPase from rabbit kidney outer medulla with the gamma-35S labeled thio-analogue of ATP in the presence of Na+ + Mg2+ and the absence of K+ leads to thiophosphorylation of the enzyme. The Km value for [gamma-S]ATP is 2.2 microM and for Na+ 4.2 mM at 22 degrees C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient nH = 2.6. (2) The thio-analogue (Km = 35 microM) can also support overall (Na+ + K+)-ATPase activity, but Vmax at 37 degrees C is only 1.13 mumol X (mg protein)-1 X h-1 or 0.09% of the specific activity for ATP (Km = 0.43 mM). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0 degree C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5-6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s-1 vs. 180 s-1), spontaneous dethiophosphorylation (0.04 s-1 vs. 0.5 s-1) and K+-stimulated dethiophosphorylation (0.54 s-1 vs. 230 s-1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s-1, Km ADP = 48 microM at 0.1 mM ATP) and is relatively K+-insensitive. The Km for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75-90% in the ADP-sensitive E1-conformation.
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PMID:Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate. 633 7

An acid slab gel electrophoresis method of high-resolving power allows detection of a phosphorylated form in the purified ATPase of the yeast Schizosaccharomyces pombe and identification of this catalytic intermediate among the different phosphopeptides of a plasma membrane preparation. At a maximum steady state rate of MgATP hydrolysis by the membrane-bound ATPase, 20 to 40% of the ATPase subunits of 100,000 daltons are in a phosphorylated form, while only 0.8% of the subunits of the purified ATPase are phosphorylated under the same conditions. The phosphorylated intermediate reaches the steady state level in less than 2 s and rapidly turns over. The phosphorylated substance is cleaved by hydroxylamine and is relatively stable in acids but is readily hydrolyzed in alkaline or in acid alcoholic media. These results suggest that the intermediate is an acylphosphate. The phosphorylation reaction has an apparent Km value of 3.0 mM MgATP for the plasma membrane-bound ATPase and 0.6 mM MgATP for the purified ATPase. Plasma membranes contain several other minor phosphorylated components whose kinetic behavior is typical of phosphorylation by protein kinase. Artifactual production of two forms of the ATPase by phenylmethanesulfonyl fluoride-sensitive proteases liberated during cell disruption is also demonstrated.
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PMID:The purified plasma membrane ATPase of the yeast Schizosaccharomyces pombe forms a phosphorylated intermediate. 644 1


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