Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol.
A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a Mr of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a Mr of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 mumol/mg protein/min, and Ka values of 67 nM and 1.1 microM for calmodulin and Ca2+, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca2+, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002645:Chickens; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006006:Glycogen Synthase / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D014336:Troponin / Q000378:metabolism; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D006006:Glycogen Synthase; D011494:Protein Kinases; D014336:Troponin; D000099211:Phospholamban