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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.
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PMID:Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms. 315 59

The mechanism by which calmodulin stimulates Ca2+ transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum (SR) was investigated. Under incubation conditions in which the majority of the phosphoprotein formed was Ca2+-dependent and no phospholamban phosphorylation was observed (10 degrees C, 15-sec incubations in the presence of 2 microM ATP), calmodulin was found to have no effect on the steady-state level of the acylphosphate phosphorylation site of Ca2+-ATPase. A significant stimulation of Mg2+, Ca2+-ATPase activity by calmodulin and a 3-fold increase in the turnover of the Ca2+ pump were, however, observed. As the ATP concentration in the incubation media was elevated (20 and 200 microM ATP), a significant degree of phosphoprotein formed was observed to be cyclic AMP (cAMP)-dependent. The degree of Ca2+-dependent phosphorylation remained constant. Under these conditions, calmodulin had no effect on the degree of phosphoprotein formed. However, when the experiments were conducted at 30 degrees C for 5 min in the presence of 500 microM ATP, a significant amount of the phosphoprotein formed was calcium-calmodulin-dependent and was additive to phosphoprotein formation observed in the presence of cAMP-dependent protein kinase. The ratio of calcium-calmodulin-dependent to cAMP-dependent phosphorylation was 1:1. K+ (110 mM) decreased the levels of phosphorylation observed in the presence of calcium and calmodulin, but had less of an effect on the levels observed in the presence of cAMP-dependent protein kinase. Autoradiographic analysis of SR membranes labeled with [32P]-ATP revealed two protein bands (24,500 and 40,000 daltons) phosphorylated in the presence of added calcium and calmodulin that were not observed in the absence of either of these additions to the reaction media. These results suggest that calmodulin stimulates Ca2+ transport by a direct effect on the Mg2+, Ca2+-ATPase. An indirect effect on Ca2+ transport via a calcium-calmodulin-dependent protein kinase, though, cannot be ruled out.
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PMID:Characterization of calmodulin-dependent and cyclic-AMP-dependent protein kinase stimulation of cardiac sarcoplasmic reticulum calcium transport. 315 44

Phospholamban, the putative protein regulator of the Ca2+ pump of cardiac sarcoplasmic reticulum, was purified to apparent homogeneity from canine cardiac sarcoplasmic reticulum vesicles by selective extraction with sodium cholate, followed by adsorption to calcium oxalate, solubilization in Zwittergent 3-14, and specific elution from p-hydroxymercuribenzoate-agarose. Phospholamban, isolated in the dephosphorylated state, was purified 80-fold in 15% yield (approximately 2 mg of phospholamban/g of sarcoplasmic reticulum protein). Nondissociated phospholamban exhibited an apparent Mr = 25,000 in sodium dodecyl sulfate-polyacrylamide gels. Partially dissociated phospholamban, induced by boiling in sodium dodecyl sulfate, exhibited five distinct mobility forms in sodium dodecyl sulfate-polyacrylamide gels, of apparent molecular weights between 5,000-6,000 and 25,000. Phospholamban was phosphorylated to a level of 190 nmol of Pi/mg of protein by cAMP-dependent protein kinase, consistent by minimum stoichiometry with a subunit molecular weight of approximately 5,000. Phospholamban prepared by the present method was different in several respects from the proteins that have been isolated in other laboratories. Pure phospholamban was cysteine rich, containing 6 residues/100 amino acid residues. Dephosphorylated phospholamban was strongly basic with a pI = 10; phosphorylation decreased the pI to approximately 6.7. Pure phospholamban (and phospholamban present in sarcoplasmic reticulum vesicles) was not readily extracted into acidified chloroform/methanol, suggesting that the protein does not behave as an acidic proteolipid. The purified protein was highly antigenic. Phospholamban was localized by immunochemical methods to cardiac membranes enriched in sarcoplasmic reticulum, but was absent from sarcoplasmic reticulum membranes prepared from fast skeletal muscle. The method described for isolation of cardiac phospholamban is highly reproducible and relatively simple, and should be useful for further detailed studies designed to probe the molecular structure of the protein.
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PMID:Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. 315 60

The effect of cAMP-dependent protein kinase on calcium uptake and protein phosphorylation in bovine aortic microsomes was examined. Acid gel electrophoresis demonstrated that the aortic microsomes contained a Ca2+-dependent, hydroxylamine-sensitive phosphoenzyme (Mr 110 kDa), characteristic of the calcium pump in sarcoplasmic reticulum, but showed no evidence of a sarcolemmal calcium pump. Calcium uptake by these aortic vesicles was markedly stimulated by oxalate, whereas calcium uptake by canine cardiac sarcolemmal vesicles was oxalate-independent. Both cAMP plus protein kinase (cAMP-PK) and catalytic subunit of protein kinase stimulated oxalate-supported calcium uptake by bovine aortic microsomes 23 +/- 3% (P less than 0.05) at 0.3 microM Ca2+, but had no effect at 6 to 10 microM Ca2+. Catalytic subunit of protein kinase and cAMP-PK phosphorylated an 11 kDa protein in bovine aortic microsomes which comigrated with canine cardiac phospholamban after boiling in sodium dodecylsulfate. The stoichiometry of the aortic 11 kDa phosphoprotein to 110 kDa phosphoenzyme was approximately 1:1. These data are consistent with the recent identification of phospholamban in various smooth muscles, and suggest that cAMP-mediated vascular relaxation may in part be attributable to stimulation of calcium uptake by the sarcoplasmic reticulum.
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PMID:Regulation of calcium uptake in bovine aortic sarcoplasmic reticulum by cyclic AMP-dependent protein kinase. 322 9

In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO4. In the absence of NaDodSO4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.
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PMID:Partial purification and characterization of thrombolamban, a 22,000 dalton cAMP-dependent protein kinase substrate in platelets. 342 96

Phospholamban was purified by two different preparation protocols. The products of both preparations are immunochemical similar. They differ, however, with regard to their phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. It is suggested that the lipid environment plays a crucial role in the exposition of the phosphorylation sites of phospholamban.
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PMID:Differences in phosphorylation of isolated phospholamban in dependence on the preparation procedure. 343 6

Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of which remains to be defined. The aim of this study was therefore to characterize the latter kinase, called phospholamban kinase. Phospholamban kinase was purified approximately 42-fold with a yield of 11%. The purified fraction exhibits a specific activity of 6.5 nmol of phosphate incorporated into exogenous phospholamban per minute per milligram of protein. Phospholamban kinase appears to be a high molecular weight enzyme and presents a broad substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin regulatory light chain being the best substrates. Phospholamban kinase phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data suggest that phospholamban kinase might be an isoenzyme of the multifunctional Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration velocity of the protein. In addition, this protein that binds calmodulin was always found to be present in fractions containing phospholamban kinase activity. This Mr 56 000 protein is therefore a good candidate for being a subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two distinct entities.
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PMID:Characterization and partial purification of cardiac sarcoplasmic reticulum phospholamban kinase. 373 Mar 67

Phospholamban, a putative regulator of the Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum (SR), was purified from canine cardiac SR membranes. Cardiac SR was extracted with deoxycholate and fractionated with ammonium sulfate followed by gel permeation high performance liquid chromatography in the presence of the nonionic detergent, octa-ethylene glycol mono-n-dodecyl ether (C12E8), and KI. Further purification was achieved with CM-Sepharose CL 6B column chromatography in the presence of C12E8. The purified phospholamban showed a single band of 22,000 daltons on neutral sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412) and 27,000 daltons on alkaline SDS gels (Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685). Boiling of phospholamban in 2% SDS produced total conversion into the lower molecular weight component on SDS gels (11,000 on Laemmli gel and 10,500 on Weber and Osborn gel). The apparent molecular weight of phospholamban on SDS gels was slightly increased by cAMP-dependent phosphorylation. The extent of phosphorylation catalyzed by cAMP-dependent protein kinase in the purified phospholamban preparations was about 42 nmol of phosphate/mg of protein when the protein concentration was determined by the method of Lowry et al. (Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275), or 138 nmol/mg of protein based on the protein concentration estimated by the dye absorption method. Rabbit antisera were prepared against purified phospholamban. The obtained antisera were found to bind to purified phospholamban as well as that in cardiac SR. No reaction was detected in fast skeletal muscle SR by immunofluorescent staining of Western blots. The present preparation of purified phospholamban and the antisera should facilitate further understanding of the regulatory action of phospholamban on the calcium pump ATPase.
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PMID:Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. 388 14

The phosphorylation-induced mobility shift of the high molecular weight form of phospholamban (24,500 daltons) in the cardiac sarcoplasmic reticulum produced on 3',5'-cyclic AMP (cAMP)-dependent phosphorylation with 5 mM ATP was resolved into five clear steps on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and on Ca2+-calmodulin-dependent phosphorylation into ten steps. The mobility shift of the low molecular weight form of phospholamban (less than 14,400 daltons) in these reactions occurred in one step and two steps, respectively. With the two protein kinase activities, the electrophoretic pattern of the mobility shifts of the high and low molecular weight forms of phospholamban was similar to that obtained with Ca2+-calmodulin-dependent protein kinase alone. The results of pulse-chase experiments involving the centrifuge column method suggested that the site(s) of phosphorylation by cAMP- and Ca2+-calmodulin-dependent protein kinase activities are on the same phospholamban molecule. Two-dimensional tryptic peptide maps of phosphorylated phospholamban indicated that cAMP-dependent protein kinase phosphorylates at a single site, A, and Ca2+-calmodulin-dependent protein kinase phosphorylates at sites C1 and C2 in the low molecular weight form, where A is different from C1 but may be the same as C2. The high molecular weight form of phospholamban is suggested to be a pentamer of identical monomers (low molecular weight form) having one phosphorylation site for cAMP-dependent protein kinase and two for Ca2+-calmodulin-dependent protein kinase.
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PMID:Subunit structure and multiple phosphorylation sites of phospholamban. 395 97

Muscarinic agonists inhibit cyclic AMP (cAMP)-induced phosphorylation of the cardiac protein phospholamban. The mechanism of this muscarinic inhibition of phosphorylation of phospholamban appears to occur at more than one level in the series of reactions comprising the adenylate cyclase, cAMP-dependent protein kinase system. Muscarinic agonists attenuate hormone and drug stimulation of cardiac adenylate cyclase. This results in reduced tissue levels of cAMP and diminished phosphorylation of cardiac proteins and consequent inhibition of biochemical and inotropic effects of drugs that act via cAMP. The mechanism of muscarinic inhibition of adenylate cyclase is only partially understood, but probably involves the inhibitory guanine nucleotide-binding regulatory protein. In addition to the inhibition of adenylate cyclase, muscarinic agonists appear to be able to inhibit the effects of cAMP. The mechanism for this second effect of muscarinic agonists is unknown.
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PMID:Mechanisms of muscarinic modulation of protein phosphorylation in intact ventricles. 608 11


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