Cyclic Adenosine 3′,5′-Monophosphate-stimulated Protein Kinase and a Substrate Associated with Cardiac Sarcoplasmic Reticulum

Abstract A cyclic adenosine 3',5'-monophosphate (cAMP)-stimulated protein kinase and a substrate have been demonstrated in a morphologically and enzymatically defined sarcoplasmic reticulum preparation from canine heart. The self-phosphorylation of these vesicles was stimulated 2.5- to 3-fold by cAMP with half-maximal stimulation at 8.5 x 10-8 m. The kinase activity phosphorylated histones and required a divalent cation, magnesium. With an optimal concentration of MgCl2 (15 mm), millimolar concentrations of calcium inhibited the enzyme. The apparent Km values for ATP in the presence and in the absence of cAMP were 2.0 x 10-5 m and 3.3 x 10-5 m, respectively. The self-phosphorylation of the sarcoplasmic reticulum was not inhibited by the specific heat-stable protein inhibitor of cAMP-stimulated protein kinases and not maximally activated by cyclic guanosine 3',5'-monophosphate. The phosphorylated membrane substrate had the characteristics of a protein with a phosphoester bond. The reported cAMP-stimulated calcium uptake of cardiac sarcoplasmic reticulum may be mediated by this kinase and its phosphorylated substrate.