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)

Properties of (Ca2+ + Mg2+) adenosine triphosphatase (ATPase) in plasma membranes from boar epididymal spermatozoa are described. Enzyme activity is optimum at high pH and has a high affinity for Ca2+. It is not inhibited by the calmodulin antagonist trifluoperazine (TFP), but it is inhibited by low concentrations of Ca2+. Plasma membrane vesicles obtained by hypotonic lysis of intact sperm [mixed inside-out (IOV) and right side-out (ROV) vesicles] transport 45Ca2+ in the presence of oxalate. Similar to the Ca2+-stimulated Mg ATPase activity, transport is unaffected by TFP, but unlike the ATPase, transport is at an optimum rate near neutral pH and is completely inhibited by p-chloromercurphenylsulfonate (pCMS). When plasma membranes are labeled in the presence and absence of Ca2+ and Mg2+ with [gamma-32P]ATP, differences in the intensity of labeling and lability of bound 32P to alkali and hydroxylamine suggest that two polypeptides between 100-120K may be related to a transport ATPase. The addition of TFP at concentrations which stimulate net Ca2+ uptake in intact cells causes intense labeling of a single neutrally charged protein near 68K. These labeling patterns and the properties of (Ca2+ + Mg2+) ATPase identify particular plasma membrane proteins (PMPs) from the complex surface of these cells that may be involved in Ca2+-dependent functions and support the view that calmodulin is not directly involved in the regulation of ATP-driven Ca2+ efflux from boar spermatozoa.
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PMID:Characterization of (Ca2+ + Mg2+) adenosine triphosphatase activity and calcium transport in boar sperm plasma membrane vesicles and their relation to phosphorylation of plasma membrane proteins. 615 5

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.
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PMID:Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase. 615 48

(Ca2+ + Mg2+)-dependent ATPase purified from sarcoplasmic reticulum was phosphorylated by [gamma-32P]ATP in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, dimethyl sulfoxide, and magnesium up to levels of 1.0 mumol of phosphoenzyme/g of protein. In the presence of 30% (v/v) dimethyl sulfoxide, phosphorylation by ATP measured in the absence of calcium was abolished by 100 mM NaCl or KCl. Optimal pH for phosphoenzyme formation was 5.5 to 6.0. The phosphoenzyme levels were not significantly affected by varying the temperature of the reaction medium in the range from 0 to 20 degrees C. Increasing dimethyl sulfoxide concentrations lead to a progressive increase of the steady state level of phosphoenzyme and to a decrease of both the velocities of formation and breakdown of phosphoenzyme. The observed decrease of phosphoenzyme hydrolysis was greater than that of formation, accounting for the higher phosphoenzyme levels observed with increasing dimethyl sulfoxide concentrations. Phosphoenzyme formed from ATP in the absence of calcium is an acylphosphate-type compound, as demonstrated by its pH and hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the (Ca2+ + Mg2+)-ATPase as shown by polyacrylamide gel electrophoresis. Controls were performed that eliminate both the possibility of calcium bound to the enzyme and the possibility of enzyme phosphorylation by contaminant 32Pi.
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PMID:Phosphorylation by ATP of sarcoplasmic reticulum ATPase in the absence of calcium. 621 9

The spontaneous contractions of cultured chick skeletal muscle fibers were abolished by growth of cultures in the presence of tetrodotoxin (TTX). Inhibition of the contractile activity of cultured myofibers was associated with a marked reduction in the rate of azide-insensitive, ATP-dependent Ca2+ uptake by the total particulate fraction of cell homogenates and by purified sarcoplasmic reticulum. Myosin heavy chain (MHC) accumulation and azide-insensitive, ATP-dependent Ca2+ uptake into a total cell membrane fraction were measured simultaneously in the same culture dish. A decrease in the activity of the ATP-dependent Ca2+ uptake system preceded a significant reduction in MHC content of contraction-inhibited cultures. The reduced rate of Ca2+ uptake observed in the sarcoplasmic reticulum from TTX-treated cultures paralleled a decrease in the amount of enzymatically active Ca2+-transport ATPase. The cellular concentration of the ATPase was estimated from a measurement of the concentration of the Ca2+-dependent, hydroxylamine-sensitive, steady state level of phosphorylated intermediate formed in culture microsomes. In contrast to the changes observed in activity of the sarcoplasmic reticulum ATPase and MHC content of TTX-treated cultures, neither the specific activity of creatine kinase nor the accumulation of the MM isoenzyme were affected. It is therefore concluded that the contractile activity of muscle has a selective effect on the maintenance of the adult skeletal muscle phenotype.
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PMID:Effect of tetrodotoxin relaxation of cultured skeletal muscle on the sarcoplasmic reticulum Ca2+-transport ATPase. 622 Sep 16

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


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