Control of phosphorylase in cultured rat skeletal muscle cells. Changes in synthesis and degradation resulting from differentiation and muscle activity.
The control of phosphorylase levels was investigated in rat skeletal muscle cells developing in vitro. The amount of enzyme was directly measured after immunoprecipitation using specific antibodies. The rate of phosphorylase synthesis was estimated by measuring the initial rate of formation of [3H]phosphorylase after incubating cells with [3H]tyrosine. Rates of degradation were determined either from pulse-chase experiments using [3H]tyrosine or by the loss of enzymatic activity following inhibition of protein synthesis. A large increase in phosphorylase occurred at the time myoblasts were fusing into myotubes. The accumulation of enzyme was preceded by a marked increase in the synthetic rate and was associated with a severalfold increase in the half-life of the enzyme. Following fusion, the myotubes began to spontaneously contract, and shortly thereafter, decreases in both the half-life and amount of phosphorylase were observed. The paralytic agents tetrodotoxin and lidocaine were without effect on phosphorylase levels before the onset of spontaneous activity; however, both agents increased the amount of enzyme when added to contracting myotubes. Tetrodotoxin had little effect on synthesis of [3H]phosphorylase but doubled the half-life of the enzyme. These and other results indicate that the increase in phosphorylase in differentiating muscle cells results from the coordinate control of both its synthesis and degradation, and that muscle activity decreases phosphorylase by increasing its degradation.
D000818:Animals; D000936:Antigen-Antibody Complex; D002454:Cell Differentiation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003513:Cycloheximide / Q000494:pharmacology; D007106:Immune Sera / Q000302:isolation & purification; D007700:Kinetics; D008012:Lidocaine / Q000494:pharmacology; D009132:Muscles / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D010763:Phosphorylase b; D006005:Phosphorylases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013779:Tetrodotoxin / Q000494:pharmacology
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D013779:Tetrodotoxin; D003513:Cycloheximide; D008012:Lidocaine; D010763:Phosphorylase b; D006005:Phosphorylases