Abstract
Studies of rat skeletal glycogen metabolism carried out in a perfused hindlimb system indicated that epinephrine activates phosphorylase via the cascade of phosphorylation reactions classically linked to the beta-adrenergic receptor/adenylate cyclase system. The beta blocker propranolol completely blocked the effects of epinephrine on cAMP, cAMP-dependent protein kinase, phosphorylase, and glucose-6-P, whereas the alpha blocker phentolamine was totally ineffective. Omission of glucose from the perfusion medium did not modify the effects of epinephrine. Glycogen synthase activity in control perfused and nonperfused muscle was largely glucose-6-P-dependent (-glucose-6-P/+glucose-6-P activity ratios of 0.1 and 0.2, respectively). Epinephrine perfusion caused a small decrease in the enzyme's activity ratio (0.1 to 0.05) and a large increase in its Ka for glucose-6-P (0.3 to 1.5 mM). This increase in glucose-6-P dependency correlated in time with protein kinase activation and was totally blocked by propranolol and unaffected by phentolamine. Comparison of the kinetics of glycogen synthase in extracts of control and epinephrine-perfused muscle with the kinetics of purified rat skeletal muscle glycogen synthase a phosphorylated to various degrees by cAMP-dependent protein kinase indicated that the enzyme was already substantially phosphorylated in control muscle and that epinephrine treatment caused further phosphorylation of synthase, presumably via cAMP-dependent protein kinase. These data provide a basis for speculation about in vivo regulation of the enzyme.
Highlights
Studies of rat skeletal muscle glycogen metabolism Several groups [1,2,3] working with purified preparations of carried out in aperfusedhindlimb system indicated rabbit skeletalmuscle glycogen synthase, have recently shown that epinephrine activates phosphorylase via the cas- that measurementof the enzyme’s K,’ for glucose-6-P is more cade of phosphorylationreactions classically linked to sensitive than the-glucose-6-P/+glucose-6-P activity ratio as the/%adrenergic receptor/adenylate cyclase system. an index of its phosphorylation state [1,3]
Since the various forms of glycogen synthase can be distinguishedby their sensitivity to activation byglucose-6-P as well as by their (-glucose-6-P/+glucose-6-P) activity ratio, we studied theeffects of different concentrations of glucose-6
To determinewhetherthechanges in the sensitivity of glycogen synthase toglucose-6-P induced by epinephrine were mediated by the p receptor-CAMP-linked phosphorylation system, theeffects of adrenergic blockers were examined.Fig. 5 shows that propranolol prevented the shift in thgelucose-6P sensitivity curve causedby epinephrine at 10 min, whereas phentolamine had no effect
Summary
ComparThis increase in glucose-6-Pdependency correlated in ison of perfusion data with observations from phosphorylation timewithprotein kinase activation and was totally studies onpurified rat skeletalmuscle glycogen synthase and blockedbypropranololandunaffectedbyphentolother data [1,2,3,4, 19] have allowed us to speculate on the in amine. Thesfeiltered supernatants were used initially to assess the effects of glucose-6-P concentration onglycogen synthase activity and to estimate a K, for the enzyme by utilizing. CAMP-dependent protein kinase was assayed according tothe method of Keely and Corbin [23]. Partial Purification and Phosphorylation of Rat SkeletaMl uscle GlycogenSynthase-Glycogen synthase a' was purified from rat hindlimb skeletal muscle essentially by the procedure described for the rabbit muscle enzyme [4]. Glucosamine-6-P-Sepharosewas prepared according to the method of Miller et al [35]
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