Rat skeletal muscle phosphorylase kinase: turnover and control of isozyme levels in culture.

The expression of phosphorylase kinase was investigated in rat skeletal muscle cells developing in vitro. The enzyme was immunoprecipitated from cells cultured in the presence of [35S]methionine, and the 35S-labeled alpha-, alpha'-, and beta-subunits of the kinase were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Fusion of myoblasts into myotubes was associated with marked increases in the amounts of kinase activity and the three 35S-labeled subunits. In 2-wk-old myotubes, the net amount of alpha'-subunit represented less than 20% of the total alpha-subunits (alpha + alpha'); however, alpha'-subunits appeared to be synthesized at least as rapidly as alpha-subunits. That alpha'-subunits were degraded more rapidly was confirmed by pulse-chase experiments, which also indicated that alpha'-subunits were not formed by proteolytic processing of the larger alpha-subunit. Inhibition of the spontaneous contractile activity of the myotubes with lidocaine markedly increased both phosphorylase kinase activity and the amounts of the 35S-labeled subunits. The divalent cation ionophore, A23187, decreased the alpha-subunits by 60%, but did not change levels of the alpha'-subunits. Taken together, the present results indicate that rat myotubes synthesize the two isozymes of phosphorylase kinase, and that levels of both are controlled by differentiation and muscle activity.