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)

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.
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PMID:Calcium regulation by lens plasma membrane vesicles. 284 Aug 57

Phospholamban, the putative regulator for the calcium pump, was purified to apparent homogeneity and in high yields from canine cardiac sarcoplasmic reticulum membranes. Purified phospholamban migrated with an apparent Mr of 27,000 in alkaline sodium dodecyl sulfate-polyacrylamide gels, and upon boiling in 7.5% sodium dodecyl sulfate, it dissociated into a lower molecular weight component of 5500-6000. Purified phospholamban contained 0.62 +/- 0.09 mumol of lipid Pi/mg of protein, and the major phospholipids were phosphatidylserine (34%), phosphatidylcholine (22%), sphingomyelin (17%), phosphatidylinositol (13%), and phosphatidylethanolamine (9%). Phospholamban was phosphorylated by cAMP-dependent protein kinase to a level of 207 nmol of Pi/mg, and this would indicate an incorporation of 1 mol of phosphate/mol of protein, assuming a molecular weight of 5500 for phospholamban. Phosphorylation of phospholamban could be reversed by a "phospholamban phosphatase" isolated from canine cardiac cytosol. Phospholipids associated with the purified phospholamban were also phosphorylated in the presence of the catalytic subunit of cAMP-dependent protein kinase, and the maximal phosphate incorporation was 4 nmol/mg of protein. The main phospholipids phosphorylated were phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate. Phosphorylation of phospholipids was inhibited by the heat-stable inhibitor protein of the cAMP-dependent protein kinase, and it could be also reversed by the phospholamban phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation and dephosphorylation of purified phospholamban and associated phosphatidylinositides. 284 74

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

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

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

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP) dependent protein kinase on a 22 000-dalton protein. Phosphorylation enhances the initial rate of Ca2+ uptake and Ca2+-ATPase activity. To determine the molecular mechanism by which phosphorylation regulates the calcium pump in SR, we examined the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Cardiac sarcoplasmic reticulum was preincubated with cAMP and cAMP-dependent protein kinse in the presence (phosphorylated SR) and absence (control) of adenosine 5'-triphosphate (ATP). Control and phosphorylated SR were subsequently assayed for formation (4-200 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase in media containing 100 microM [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E approximately P formed at low free [Ca2+] (less than or equal to 7 microM), but the effect was less at high free Ca2+ (greater than or equal to 10 microM). This stimulation was associated with a decrease in the dissociation constant for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E approximately P formation of calcium-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E approximately P decomposition. These findings indicate that phosphorylation of cardiac SR by cAMP-dependent protein kinase regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the calcium pump observed at steady state.
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PMID:Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase. 625 93

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.
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PMID:Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase. 630 13

Stimulation of secretion in exocrine cells by agonists involving cAMP as second messenger is associated with the phosphorylation of a specific membrane-associated 22.4-kDa protein (protein III) (Jahn et al.). Here it is shown by subcellular fractionation of rat parotid gland lobules that protein III is associated with the endoplasmic reticulum. The submicrosomal fractions containing protein III, also contain the ATP-dependent microsomal calcium pump activity. Protein III in microsomal subfractions can be phosphorylated in vitro with catalytic subunit from cAMP-dependent protein kinase. Phosphorylated protein III contains exclusively P-serine. Protein III can be removed from ER-membranes with acid chloroform-methanol or Triton X-114, but not by high salt wash indicating that it is tightly associated with the membranes. Protein III is smaller than phospholamban and, in contrast to phospholamban, resistant to heating in SDS. A relationship between phosphorylation of protein III and microsomal calcium sequestration is discussed.
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PMID:Specific phosphorylation of a protein in calcium accumulating endoplasmic reticulum from rat parotid glands following stimulation by agonists involving cAMP as second messenger. 631 93

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.
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PMID:Calcium-calmodulin-dependent phosphorylations in the control of muscular contraction? 701 31

The underlying mechanism of Ca2+ uptake function of cardiac sarcoplasmic reticulum (SR) was investigated in the rat septic shock model produced by cecal ligation and puncture (CLP). The results are as follows. During the early phase of sepsis, the initial rate of ATP-dependent Ca2+ uptake by SR was decreased, while both the capacity of Ca2+ uptake and the activity of Ca(2+)-ATPase were unaffected. In the late sepsis, the impairment in SR function was even greater as the initial rate and the capacity of Ca2+ uptake by SR were significantly decreased, and this was paralleled by a reduction in Ca(2+)-ATPase activity. Although Ca2+ affinity (Km value) to calcium pump and the A0.5 values for Mg2+ and ATP activation on the Ca2+ uptake rate were unchanged, during sepsis the phosphorylation of SR vesicles by adding of catalytic subunit of the cAMP-dependent protein kinase (PKA), calmodulin, or the fragment of PKC into Ca2+ uptake buffer, failed to stimulate Ca2+ uptake activities of SR isolated from early or late septic rats. These data suggest that depression of cardiac SR function is aggravated as sepsis develops, the impairment of SR Ca2+ uptake is possibly based on a mechanism of defective phosphorylation of SR rather than the ionic and energic regulatory actions of Ca2+, Mg2+, ATP on cardiac SR.
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PMID:[Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism during rat septic shock]. 748 74


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