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)

Phosphorylation induced by cAMP-dependent protein kinase was examined in a plasma membrane-enriched fraction from control and beta-adrenergic-stimulated rat aortic myocytes. Phosphorylation of a 16 kDa protein which copurified with the plasma membrane marker (Na+ + K+)-ATPase was most prominent. It was decreased by pretreatment of the myocytes with isoproterenol and the effect of isoproterenol was inhibited by propranolol. Both phosphorylation induced by cAMP-dependent protein kinase and its inhibition by isoproterenol pretreatment declined in preparations exposed to endogenous phosphatase. These results provide strong evidence that beta-adrenergic stimulation of aortic myocytes induces in situ phosphorylation of a 16 kDa plasma membrane protein.
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PMID:Cyclic AMP-dependent phosphorylation of a 16 kDa protein in a plasma membrane-enriched fraction of rat aortic myocytes. 406 43

Fructose 2,6-bisphosphate is a potent allosteric activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. It potentiates the effect of AMP on both enzymes. A great deal of compelling evidence supports the hypothesis that fructose 2,6-bisphosphate plays a key role in the hormonal and substrate regulation of substrate cycling at the fructose 6-phosphate/fructose 1,6-bisphosphate level in liver. This regulation is exerted at the level of the enzyme activities responsible for the synthesis and degradation of fructose 2,6-bisphosphate. Synthesis of the compound is catalyzed by a unique enzyme which transfers the gamma-phosphate of ATP to the C2 position of fructose 6-phosphate (ATP:D fructose 6-phosphate 2-phosphotransferase) while degradation is catalyzed by a phosphohydrolase activity which is specific for the C-2 position of fructose 2,6-bisphosphate (D-fructose 2,6-bisphosphate 2-phosphohydrolase). These activities are distinct from the classical 6-phosphofructo 1-kinase and fructose 1,6-bisphosphatase with regard to molecular weight, interaction with ligands, and the efficiency with which phosphoryl transfer occurs. Both activities have been purified to homogeneity and have been shown to be present in a single enzyme protein, i.e. the enzyme is bifunctional. Incubation of the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase with cAMP-dependent protein kinase and ATP leads to phosphorylation of the enzyme resulting in inactivation of the phosphotransferase activity and stimulation of the phosphohydrolase activity. Since fructose 2,6-bisphosphate is not further metabolized and can only be recycled to fructose 6-phosphate, simultaneous modulation of the synthesis and degradation of the compound by covalent modification of a single protein provides a very efficient and sensitive regulatory mechanism. The bifunctional enzyme was also shown to possess an ATPase activity which was nearly equal to the activity of the kinase reaction. However, in the presence of fructose 6-phosphate the enzyme did not transfer phosphate to water but rather to the C-2 position of the phosphorylated sugar. The ability of the enzyme to catalyze a partial reaction at a rate nearly equal to that of the forward reaction suggested that the reaction mechanism of the kinase proceeds by a two step transfer, i.e. via a phosphoryl enzyme intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a unique bifunctional enzyme. 610 May 82

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.
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PMID:Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum. 612 98

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.
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PMID:Regulation of calcium accumulation and efflux from platelet vesicles. Possible role for cyclic-AMP-dependent phosphorylation and calmodulin. 613 52

R24571 a derivative of the antimycotic miconazole, appears to be 5 to 8 times more potent than trifluoperazine in its ability to inhibit the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum vesicles. The cAMP-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum was not affected by R24571. Sarcoplasmic reticulum Ca-dependent ATPase phosphoprotein intermediate formation was inhibited by R24571 concentrations that were 20 to 30 times greater than those required to inhibit calmodulin-dependent phosphorylation. However, both Ca-dependent and independent ATPase activities, as well as calcium uptake, were inhibited by R24571 concentrations that were similar to, or less than, those concentrations required to inhibit calmodulin-dependent sarcoplasmic reticulum phosphorylation. These results indicate the caution that should be exercised in using this new compound in assessing the possible involvement of calmodulin in other membrane processes.
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PMID:Inhibition of calmodulin-dependent and independent cardiac sarcoplasmic reticulum activities by R24571. 613 10

Binding of both synthetic poly(A) and naturally occurring poly(A) (+)mRNA as well as DNA to microtubule protein is mediated by microtubule-associated proteins; tubulin itself is not capable of binding these polymers. Bovine brain microtubule protein from immature animals was found to have a significantly lower capacity to bind poly(A) than microtubule protein from old animals. On the other hand, "old" microtubule protein binds DNA more efficiently than "immature" microtubule protein. Microtubule-associated protein 2 [preferred binding site for DNA] and tau proteins [preferred binding site for poly (A)] are specifically phosphorylated by a microtubule-associated, cAMP-dependent protein kinase. It was found that the affinity of microtubule protein for poly(A) is markedly decreased by autophosphorylation of the protein; in the case of DNA, the decrease in affinity was less. Autophosphorylation of "immature" microtubule proteins diminished the binding capacity for poly(A) to a greater extent than do "old" proteins. Scatchard plot analysis revealed that microtubule-protein possesses two different binding sites for poly(A). The corresponding dissociation constants were found to be increased in the phosphorylated system, but phosphorylation does not appear to alter the total number of binding sites. Compared to immature animals, microtubule protein from "old" bovine brains was found to have a reduced number of binding sites for poly(A), whereas the values of the dissociation constants remain unchanged. In contrast to total microtubule protein and homogeneous microtubule-associated protein 2, only one kind of binding site for poly(A) could be detected in homogeneous tau protein. No influence of different RNA or DNA species on microtubule protein-associated cAMP-dependent protein kinase, adenosine triphosphatase and guanosine triphosphatase activities could be detected.
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PMID:Binding of polyribonucleotides and polydeoxyribonucleotides to bovine brain microtubule protein: age-dependent modulation via phosphorylation of high-molecular-weight microtubule-associated proteins and tau proteins. 614 31

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.
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PMID:Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump. 614 39

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.
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PMID:Ca2+/calmodulin-dependent phospholamban kinase from cardiac sarcoplasmic reticulum is distinct from phosphorylase kinase and forms a regulatory complex with phospholamban and the Ca2+-ATPase. 622 Jun 53

The Ca2+-pumping ATPase has been isolated from calf heart sarcolemma by calmodulin affinity chromatography (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 3263-3270) as a polypeptide of Mr about 140,000. The purified enzyme has high affinity for Ca2+ in the presence of calmodulin (Km about 0.4 microM) but shifts to a low affinity state (Km about 20 microM) in its absence. Calmodulin increases also the Vmax of the enzyme. The effects of calmodulin are mimicked by phosphatidylserine and by a limited proteolytic treatment of the enzyme with trypsin. The purified ATPase can be reconstituted in asolectin liposomes, where it pumps Ca2+ with an approximate stoichiometry to ATP of 1. The purified (and reconstituted) enzyme is not phosphorylated by added ATP and cAMP-dependent protein kinase under conditions where the enzyme in situ is stimulated concomitant with the phosphorylation of the sarcolemmal membrane (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 9371-9373). Hence, the target of the regulatory phosphorylation system is not the ATPase molecule. The purified ATPase cross-reacts with an antibody raised against the erythrocyte Ca2+-pumping ATPase. Under the same conditions, the purified sarcoplasmic reticulum Ca2+-ATPase does not react. The proteolytic splitting pattern of the purified heart sarcolemma and erythrocyte enzymes are similar but not identical.
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PMID:Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma. 622 26

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.
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PMID:Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. 625 61


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