PHOSPHORYLATION OF PURIFIED CARDIAC-MUSCLE C-PROTEIN BY PURIFIED CAMP-DEPENDENT AND ENDOGENOUS CA-2+-CALMODULIN-DEPENDENT PROTEIN-KINASES

Abstract

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. C-protein was purified in high yield from cardiac muscle (.apprx. 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a MW 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 .apprx. 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 Ca and calmodulin (.apprx. 160 pmol of phosphate incorporated/min per mg of C-protein). This endogenous kinase catalyzes the incorporation of .apprx. 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 .mu.M, Vmax = 18.6 .mu.mol/min per 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.