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 kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.
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PMID:Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C. 215 96

Purified Na+, K(+)-ATPase was phosphorylated by [gamma-32P]ATP in a medium containing dimethylsulfoxide and 5 mM Mg2+ in the absence of Na+ and K+. Addition of K+ increased the phosphorylation levels from 0.4 nmol phosphoenzyme/mg of protein in the absence of K+ to 1.0 nmol phosphoenzyme/mg of protein in the presence of 0.5 mM K+. Higher velocities of enzyme phosphorylation were observed in the presence of 0.5 mM K+. Increasing K+ concentrations up to 100 mM lead to a progressive decrease in the phosphoenzyme (EP) levels. Control experiments, that were performed to determine the contribution to EP formation from the Pi inevitably present in the assays, showed that this contribution was of minor importance except at high (20-100 mM) KCl concentrations. The pattern of EP formation and its KCl dependence is thus characteristic for the phosphorylation of the enzyme by ATP. In the absence of Na+ and with 0.5 mM K+, optimal levels (1.0 nmol EP/mg of protein) were observed at 20-40% dimethylsulfoxide and pH 6.0 to 7.5. Addition of Na+ up to 5 mM has no effect on the phosphoenzyme level under these conditions. At 100 mM Na+ or higher the full capacity of enzyme phosphorylation (2.2 nmol EP/mg of protein) was reached. Phosphoenzyme formed from ATP in the absence of Na+ is an acylphosphate-type compound as shown by its hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the alpha-subunit of the Na+, K(+)-ATPase as demonstrated by acid polyacrylamide gel electrophoresis followed by autoradiography.
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PMID:Phosphorylation of Na+, K(+)-ATPase by ATP in the presence of K+ and dimethylsulfoxide but in the absence of Na+. 215 51

Treatment of renal Na,K-ATPase with N-acetylimidazole (NAI) results in loss of Na,K-ATPase activity. The inactivation kinetics can be described by a model in which two classes of sites are acetylated by NAI. The class I sites are rapidly reacting, the acetylation is prevented by the presence of ATP (K0.5 congruent to 8 microM), and the inactivation is reversed by incubation with hydroxylamine. These data suggest that the class I sites are tyrosine residues at the ATP binding site. The second class of sites are more slowly reacting, not protected by ATP, nor reversed by hydroxylamine treatment. These are probably lysine residues elsewhere in the protein. The associated K-stimulated p-nitrophenylphosphatase activity is inactivated by acetylation of the class II sites only; thus the tyrosine residues associated with ATP binding to the catalytic center are not essential for phosphatase activity. Inactivated enzyme no longer has high-affinity ATP binding associated with the catalytic site, although low-affinity ATP effects (inhibition of phosphatase and deocclusion of Rb) are still present. The inactivated enzyme can still be phosphorylated by Pi, occlude Rb+ ions, and undergo the major conformational transitions between the E1 Na and E2 K forms of the enzyme. Thus acetylation of the Na,K-ATPase by NAI inhibits high-affinity ATP binding to the catalytic center and produces inactivation.
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PMID:N-acetylimidazole inactivates renal Na,K-ATPase by disrupting ATP binding to the catalytic site. 216 61

To elucidate the regulation mechanisms for sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle, the preparation of the membrane fraction of porcine aorta with which the enzyme activity could be analyzed was attempted. A Ca2(+)-activated, Mg2(+)-dependent ATPase [Ca2(+)+Mg2+)-ATPase) activity with high affinity for Ca2+ (Km = 79 +/- 18 nM) was found in a sarcolemma-enriched fraction obtained from digitonin-treated microsomes that possessed the essential properties of plasma membrane (PM) Ca2(+)-pumping ATPases, as determined for the erythrocyte and cardiac muscle enzymes. The activity was stimulated by calmodulin and inhibited by low concentrations of vanadate. Saponin had a stimulatory effect on it. The existence of the PM enzyme in the membrane fraction was substantiated by the Ca2(+)-dependent, hydroxylamine sensitive phosphorylation of a 130K protein, which could be selectively enhanced by LaCl3. The enzyme activity was potentiated by either cGMP or a purified G-kinase. Purified protein kinase C potentiated the enzyme activity. However, none of these agents stimulated the activity of the enzyme purified from microsomes by calmodulin affinity chromatography. The results suggest that the sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle is regulated by these protein kinases not through phosphorylation of the enzyme itself but through phosphorylation of membrane components(s) other than the enzyme. Phosphatidylinositol phosphate was found to stimulate the enzyme, suggesting its role in mediation of the stimulatory effects of the protein kinases.
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PMID:Sarcolemmal (Ca2(+)+Mg2+)-ATPase of vascular smooth muscle and the effects of protein kinases thereupon. 216 73

Chymotryptic cleavage of the alpha-subunit of the canine kidney Na+/K(+)-ATPase in the presence of Na+ abolishes ATPase activity and yields an 83 kDa peptide from Ala 267 to the COOH-terminus. To test the proposal that E1 to E2 conformational transition is blocked in this modified enzyme, we have made a detailed comparison of its phosphorylation with that of the native enzyme by ATP. While phosphorylation of alpha is dependent on Na+ and prevented by K+, that of the 83 kDa peptide is modestly stimulated by Na+; and only this stimulation, but not the Na(+)-independent phosphorylation is inhibited by K+. Ouabain, which inhibits alpha-phosphorylation by ATP, activates Na(+)-independent phosphorylation of the 83 kDa peptide by ATP, and inhibits the Na(+)-stimulation of this process. While there is a ouabain-stimulated phosphorylation of alpha by Pi, the 83 kDa peptide is not phosphorylated by Pi with or without ouabain. In its sensitivity to ADP, and insensitivity to K+, the phosphopeptide is similar to the E1P of the native enzyme; however, the spontaneous decomposition rate of the phosphopeptide is orders of magnitude lower than that of the native EP. Na+ has no effect on the spontaneous decomposition of the phosphopeptide; but at high Na+ concentrations (K0.5 = 350 mM) the ADP sensitivity of the phosphopeptide is reduced. The phosphopeptide, like the native EP, is acid-stable, alkaline-labile, and sensitive to hydroxylamine and molybdate. The chymotrypsin-treated enzyme catalyzes an ADP-ATP exchange activity that is stimulated by Na+. The Na(+)-independent part of this exchange, unlike that of the native enzyme, is activated by ouabain. Our findings establish that (a) the phosphorylation process and its control by Na+, K+ and ouabain are autoregulated by the NH2-terminal domain of the alpha-subunit; and (b) the often repeated assumption that the primary role of this domain is in the regulation of E1-E2 transitions is not valid.
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PMID:Autoregulation of the phosphointermediate of Na+/K(+)-ATPase by the amino-terminal domain of the alpha-subunit. 217 3

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.
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PMID:Cross-linking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin. 229 47

The possibility that H+ might substitute for Na+ at Na+ sites of Na+,K+-ATPase was studied. Na+,K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+,K+-ATPase). The specific activity was 1.1 mumol Pi/mg/min at pH 5.7, whereas the specific activity of Na+,K+-ATPase was 14 mumol Pi/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+,K+-ATPase was inhibited as effectively as Na+,K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+,K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.
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PMID:Proton transport catalyzed by the sodium pump. Ouabain-sensitive ATPase activity and the phosphorylation of Na,K-ATPase in the absence of sodium ions. 242 57

The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.
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PMID:Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains. 245 28

Immunological studies were designed to study the structure of the oligomycin sensitivity conferring protein (OSCP) integrated in the mitochondrial ATPase-ATPsynthase complex. The monoclonal antibody 2B1B1 used in this study could bind as well to purified or membrane bound OSCP as shown previously by Protein A-gold immunocytochemistry and by competitive immunotitration. In this paper, it is shown that 2B1B1 can also immunoprecipitate the F0F1 complex from a Triton X-100 extract. This means that not only, 2B1B1 binds to the surface of OSCP but also that the binding of 2B1B1 did not destroy the interactions between F0 and F1 and further demonstrates the external location of the 2B1B1 binding site in the ATPase-ATPsynthase complex. This antigenic site was located on the N-terminal sequence of OSCP, between residues 1 and 72, as demonstrated after chemical cleavage of OSCP with formic acid, hydroxylamine and partial cleavage with cyanogen bromide. The proximity of Tyr and Arg to the epitope was suggested by the lack of 2B1B1 binding to iodinated OSCP and by the susceptibility of this binding to trypsin or to endoproteinase Arg-C treatments of OSCP, respectively. A more precise location of the epitope has been attempted by using the method of synthesis of overlapping octapeptides on solid support. It was found that 2 groups of octapeptides could bind 2B1B1. The first group contained in common the sequence Pro7-Pro8-Val9-Gln10-Ile11-Tyr12- and the second group of peptides contained the sequence Arg62-Ser63-Val64-Lys65. Another monoclonal antibody, AF4H7, which competes with 2B1B1, also recognized the first group of peptides. The possible involvement of these 2 fragments in the epitope localized at the surface of OSCP is discussed. In addition, secondary structure theoretical analysis predicts that these 2 domains should be in a beta-strand configuration.
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PMID:Epitope of OSCP oligomycin sensitivity conferring protein exposed at the surface of the mitochondrial ATPase-ATPsynthase complex. 247 97

The calmodulin-stimulated ATPase of maize coleoptiles is a 140,000 Mr polypeptide. In the present study, formation of a phosphorylated intermediate by the enzyme is demonstrated. Phosphorylation is sensitive to chasing with unlabelled ATP and to hydroxylamine; lanthanum enhances its intensity while calmodulin enhances phosphorylation in the presence of lanthanum but not in its absence. Ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) inhibits phosphorylation of the purified enzyme, but microsome preparations give a band of phosphorylation of 153,000 Mr in its presence. This latter phosphorylated band was not abolished after a variety of permeabilising treatments in the presence of Triton X-100; phosphorylation of the enzyme was absent when sodium deoxycholate was used as the solubilising detergent. The identity of the 153,000 Mr band is discussed.
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PMID:The calmodulin-stimulated ATPase of maize coleoptiles forms a phosphorylated intermediate. 252


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