Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart.
The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and kappa-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K0.5 = 93 +/- 5 nM), glycogen synthase (K0.5 = 127 +/- 10 nM), and kappa-casein (K0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 microM) but a low affinity for kappa-casein (21 +/- 2 microM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.
D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D011506:Proteins; D011494:Protein Kinases