Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

摘要: Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization sodium dodecyl sulfate fractionation by polyacrylamide gel electrophoresis, single microsomal component approximately 22,000 daltons was found to bind most 32P label. The labeling this increased several fold when NaF included incubation medium. No other microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts This 22,000-dalton phosphoprotein formed cyclic AMP-dependent had stability characteristics phosphoester rather than an acyl phosphate. Washing buffered KCl did not decrease amount suggesting that is associated membranes being contaminant from soluble proteins. susceptible trypsin. Brief digestion trypsin 1 M sucrose significantly affect calcium transport activity, but prevented both subsequent phosphorylation stimulation uptake kinase, modulator pump. These results are consistent previous findings (Kirchberger, M.A., Tada, M., Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Kirchberger, Repke, D.I., 6174-6180) kinase-catalyzed reticulum, further indicate occurs at low mass (22,000 daltons) which, while separable (100,000 sulfate-polyacrylamide has ability regulate reticulum.