Effects of protein kinase modulator on cAMP-and cGMP-dependent protein kinase-catalyzed phosphorylation and the rate of calcium uptake by cardiac microsomes

45 SIGNIFICANCE OF TWO CLASSES OF PHOSPHOPROTEINS IN FUNCTION OF CARDIAC SARCOPLASMIC RETICULUM: PHOSPHORYLATION OF Ca 2+ DEPENDENT ATPase AND PHOSPHOLAMBAN. M. Tada, F. Ohmori, N. Kinoshita, M. Yamada, M. Kadoma, H. Matsuo & H. Abe. The First Dept. of Med., Osaka Univ. Med. Sch., Osaka, Japan To assess the mode of interactions between Ca2+-depen dent ATPase and its putative regulatory protein phospholamban in Cardiac microsomes (CSR), CSR was phosphorylated in the presence of Ca 2+ (i to i00 DM) and cAMP and protein kinase (PK). The phosphoenzyme of ATPase (i00,000 daltons) exhibited stability characteristics of an acyl phosphate, whereas phosphorylation of phospholamban (22,000 daltons) catalyzed by PK resulted in the formation of a phosphoester. Amino acid analysis of ATPase purified by gel filtration in sodium dodecyl sulfate revealed that it contained about 45% polar and 55% nonpolar amino acids. Glutamic and aspartic acids accounted for about 22% of the total. Cysteine and histidine were present in relatively lower amounts. Purification of phospholamban by electrophoresis on polyaerylamide gel is under investigation.

Supported byMuscularDystrophy Association of America and Japan Heart Foundation. EFFECTS OF PROTEIN KINASE MODULATOR ON cAMP- AND cGMPDEPENDENT PROTEIN KINASE-CATALYZED PHOSPHORYLATION AND THE RATE OF CALCIUM UPTAKE BY CARDIAC MICROSOMES. F. Ohmori, M. Tada, N. Kinoshita, M. Kadoma, H. Matsuo, H. Sakakibara, Y. Nimura, & H. Abe. The First Dept. of Medicine, Osaka Univ. Med. School, Fukushima-ku, Osaka 553, Japan To define the role of protein kinase (PK) in the myocardial function, we prepared from bovine hearts protein kinase modulator (PKM), possessing abilities to inhibit cAMP-dependent PK (APK) and to stimulate cGMP-dependent PK (GPK), and examined whether PKM alters Ca transport by cardiac microsomes. Sephadex G-100 chromatography of PKM separated the kinase-lnhibitory and kinase-stimulatory activities into two peaks. The former consisted of a single component of MW=I3,000, while the latter exhibited several protein bands including those with MW=26,000 and 52,000. Increasing amounts of the former produced both inhibition of APK-catalyzed phosphorylation of phospholamban and decrease in the rate of Ca uptake, which had been augmented by APK. GFK and the kinase-stimulatory component of PKM failed to alter APK-induced increase in Ca uptake.

Supported by Muscular Dystrophy Association.