Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases.
C-protein, a component of the thick filament of striated muscles, becomes phosphorylated in response to beta-adrenergic receptor stimulation and dephosphorylated in response to cholinergic receptor stimulation in heart. We have purified C-protein in high yield from cardiac muscle (approximately 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a molecular weight on sodium dodecyl sulfate polyacrylamide gels of 155,000 but the native protein migrates as a globular protein of 209,000 daltons in gel filtration on Sephacryl S-300, suggesting that it is an asymmetric molecule composed of a single 155,000-dalton polypeptide. C-protein from chicken cardiac muscle has an amino acid composition similar to that of C-proteins from other muscles. The purified protein contains approximately 0.2 mol of phosphate/mol of C-protein. The purified C-protein has no endogenous protein phosphatase activity but does exhibit protein kinase activity in the presence of calcium and calmodulin (approximately 160 pmol of phosphate incorporated/min/mg of C-protein). This endogenous kinase catalyzes the incorporation of approximately 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 microM, Vmax = 18.6 mumol/min/mg). Phosphorylation by catalytic subunit of cAMP-dependent protein kinase exhibits a broad pH optimum between pH 8 and 9 and results in the incorporation of up to 3 mol of phosphate/mol of C-protein. Phosphate is incorporated into 3-5 different sites at both phosphothreonine and phosphoserine residues. The phosphorylated C-protein does not differ from unphosphorylated C-protein with regard to Stokes radius, migration on sodium dodecyl sulfate-polyacrylamide gels, or UV spectrum.
D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D002645:Chickens; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D009124:Muscle Proteins; D011494:Protein Kinases; D000099163:Myosin Binding Protein C