| Abstract: | The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with 32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (Mapp = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the 32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of 32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 microM) inactivated glycogen synthase and increased its content of 32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The larger fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased 32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of 32P] phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme. |
