CONTROL OF GLYCOGEN-SYNTHASE AND PHOSPHORYLASE BY AMYLIN IN RAT SKELETAL-MUSCLE - HORMONAL EFFECTS ON THE PHOSPHORYLATION OF PHOSPHORYLASE AND ON THE DISTRIBUTION OF PHOSPHATE IN THE SYNTHASE SUBUNIT

The effects of amylin and insulin on the phosphorylation of glycogen s ynthase and phosphorylase were investigated using rat diaphragms incub ated with P-32i. Muscles were incubated with insulin (200 nM) or amyli n (200 nM) for 30 min before extracts were prepared. The P-32 contents of the enzymes were determined after immunoprecipitation and SDS-poly acrylamide gel electrophoresis. Amylin increased both the activity rat io (-AMP/+AMP) and the P-32 content of phosphorylase by approximately 2-fold. Insulin alone was without significant effect on phosphorylase, but insulin blocked the effect of amylin on increasing the phosphoryl ation of phosphorylase. Insulin increased the glycogen synthase activi ty ratio (low glucose-6-P/high glucose-6-P) by approximately 80%. Amyl in decreased this ratio from 0.14 to 0.08 and increased the phosphoryl ation of synthase by approximately 40%. To investigate changes in phos phorylation of different sites in the synthase, the enzyme was subject ed to exhaustive proteolysis with trypsin, and P-32-labeled fragments were separated by reverse phase high performance liquid chromatography . Insulin decreased the P-32 contents of sites 3(a + b + c) and 2(a b), which appears to account for the increase in synthase activity. Am ylin increased phosphorylation of sites 1a, 1b, and 3(a + b + c), but not sites 2(a + b). With insulin plus amylin, phosphorylation of none of the sites was significantly changed. The results indicate that the effects of amylin on glycogen synthase must involve more than activati on of cAMP-dependent protein kinase, as this kinase phosphorylates sit e 2 and does not phosphorylate sites 3(a + b + c).