Effect of Myocardial Protein Kinase Modulator on Adenosine 3′: 5′-Monophosphate-Dependent Protein Kinase-Induced Stimulation of Calcium Transport by Cardiac Sarcoplasmic Reticulum1

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Abstract
We partially purified a heat-stable protein factor (protein kinase modulator) from fresh bovine heart, which exhibited abilities to inhibit cardiac muscle adenosine 3′: 5′-monophosphate (cyclic AMP)-dependent protein kinase [EC 2.7.1.37] and to stimulate pulmonary guanosine 3′: 5′-monophosphate (cyclic GMP)-dependent protein kinase [EC 2.7.1.37], and examined its effects on cyclic AMP-dependent protein kinase-catalyzed phosphorylation of cardiac microsomal protein “phospholamban” (MW=22,000), which was previously proposed to serve as a regulator of calcium transport by cardiac sarcoplasmic reticulum (Tada, M., Kirchberger, M.A., & Katz, AM. (1975) J. Biol. Chem. 250, 2640–2647). Incubation of cardiac microsomes, consisting mainly of sarcoplasmic reticulum, with cyclic AMP, protein kinase and [γ-33P]ATP in the presence of the modulator resulted in the inhibition of phosphorylation of the 22,000-dalton protein component of the microsomal membranes. When microsomes were incubated under identical conditions with unlabeled ATP, the rate of oxalate-facilitated calcium uptake, which had been stimulated by cyclic AMP-dependent protein kinase, was found to decrease with increasing amounts of protein kinase modulator. The decrease in calcium uptake was closely correlated with the inhibition of phosphorylation of the 22,000-dalton protein. These observations provide further evidence that the phosphorylation of the 22,000-dalton protein is functionally related to calcium transport by cardiac sarcoplasmic reticulum of the heart, and thus support the previously proposed control mechanism of calcium transport.