Involvement of 60-kilodalton phosphoprotein in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum.

"Heavy" sarcoplasmic reticulum vesicles loaded with 5 mM CaCl2 in the presence of protease inhibitors were phosphorylated by addition of MgATP in the presence or absence of calmodulin. The major site of phosphorylation was a 60-kDa protein. In the absence of added calmodulin, phosphorylation of the 60-kDa protein reached its maximal value (8 pmol of P/mg of membrane protein) at 1 min. In the presence of 1 microM calmodulin, a 2-fold higher level of phosphorylation (16.1 pmol of P/mg of sarcoplasmic reticulum) was reached within a shorter time (10 s). The phosphoprotein was then spontaneously dephosphorylated. The initial rate of Ca2+ release, which was induced by a Ca2+ jump and determined by stopped-flow fluorometry using chlorotetracycline, decreased upon phosphorylation, whereas it was restored upon dephosphorylation. There was good correlation between the amount of P incorporation into the 60-kDa protein and the extent of inhibition of Ca2+ release. In the presence of added calmodulin the protein kinase activity sharply increased in the [Ca2+] range of 0.2-2 microM with a concentration for half-maximal activation at 0.6 microM. On the other hand, the protein phosphatase activity was virtually independent of calmodulin and [Ca2+] in the [Ca2+] range in which protein kinase was activated. The results suggest that the calmodulin-dependent phosphorylation of the 60-kDa protein plays an important role in the regulation of Ca2+ release from sarcoplasmic reticulum.