A calmodulin-dependent protein kinase system from skeletal muscle sarcoplasmic reticulum. Phosphorylation of a 60,000-dalton protein.

The calmodulin plus Ca2+-dependent phosphorylation of a 60,000-dalton protein was detectable in muscle homogenates and in purified sarcoplasmic reticulum membranes. Phosphorylation was enhanced when purified sarcoplasmic reticulum vesicles were extracted with 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at pH 8.0 to remove endogenous calmodulin and lower the level of residual phosphorylation. Phosphorylation of the 60,000-dalton protein was stimulated by NaF, reached maximal levels of about 55 pmol/mg of protein within 90 s and was not affected by cAMP. Half-maximal stimulation of phosphorylation required 0.1 microM calmodulin and maximal phosphorylation required 0.6 microM calmodulin; in the presence of 0.6 microM calmodulin, maximal phosphorylation required 0.3 microM Ca2+. Calmodulin-dependent phosphorylation of the 60,000-dalton protein was inhibited by trifluoperazine with a Ki of 5 microM. The pH optimum of phosphorylation was below 6.0 and was inhibited over 90% at pH 8.0. The purified 60,000-dalton protein contained phosphoserine and phosphothreonine but no phosphotyrosine. Two-dimensional gel electrophoresis and immunoprecipitation showed that the 60,000-dalton phosphoprotein was not calsequestrin. Endo-beta-N-acetylglucosaminidases H and D digestion failed to alter its molecular weight, indicating that it was not a glycoprotein. A functional role for the phosphorylation system is suggested by the observations that ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid extraction, which removes endogenous calmodulin and lowers endogenous phosphorylation levels, and high pH, which inhibits phosphorylation, lead to greatly diminished Ca2+ accumulation by sarcoplasmic reticulum vesicles.