PHOSPHORYLATION AND INACTIVATION OF GLYCOGEN-SYNTHASE BY PHOSPHORYLASE-KINASE

Skeletal muscle glycogen a4-synthase (EC 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6-P-Sepharose affinity column and then on a phosphocellulose column. This preparation of glycogen synthase was tested as a substrate for purified skeletal muscle phosphorylase kinase (ATP:phosphorylase-b phosphotransferase. EC 2.7.1.38). Phosphorylase kinase (1-10 μg/ml or 0.03-0.3 μM) catalyzes rapid phosphorylation of glycogen synthase (4.5 μM) associated with conversion of the active a form to the less active b form. In the reaction, >95% of the 32P incorporation from [γ-32P]ATP goes into the synthase subunit almost exclusively in the trypsin-insensitive region which is responsible for synthase a-to-b conversion. Synthase phosphorylation or inactivation catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, is ATP dependent, is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased 10-fold by prior activation of the phosphorylase kinase with MgATP and cyclic AMP. With activated phosphorylase kinase at pH 8.2 the apparent K(m) and V(max) are approximately 70μM and 4 μmol/min per mg with glycogen synthase as substrate and 70 μM and 9 μmol/min per mg with phosphorylase as substrate. It is concluded that glycogen synthase is a substrate in vitro for phosphorylase kinase, a Ca2+-dependent enzyme. The possible physiological significance of this reaction is discussed.