Phosphorylation of a 22,000-Dalton Component of the Cardiac Sarcoplasmic Reticulum by Adenosine 3’ : S-Monophosphate-dependent Protein Kinase*

Cardiac microsomes were incubated with [y-azP]ATP and a cardiac adenosine 3’:5’-monophosphate (cyclic AMP)dependent protein kinase in the presence of ethylene glycol bis(&aminoethyl ether)-A’, IV’-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the a2P label. The a*P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of azP label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein lrinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KC1 did not decrease the amount of azP labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calciums uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings @rRCHBERGER, M. A., TADA, M., AND KATZ, A. M. (1974) J. Biol.