Yeast glycogen synthetase in the glucose 6-phosphate-dependent form. I. Purification and properties.

The glucose-6-P-dependent form of glycogen synthetase has been purified about 5,000-fold from yeast extracts. The enzyme was separated from the glucose-6-P-independent form by DEAE-cellulose chromatography and from other proteins by a two-step agarose chromatography, using complex formation with glycogen to change the elution properties of the synthetase. The purified material shows a single band on disc gel electrophoresis and possesses a molecular weight of 300,000, as measured by sedimentation equilibrium. Acrylamide electrophoresis in the presence of sodium dodecyl sulfate yields a major band, molecular weight about 77,000, and a minor band of variable intensity, with molecular weight about 71,000. The faster band is believed to be a proteolysis degradation product of the slow band. Thus, the native enzyme would consist of four subunits of qqual molecular weight. The enzyme was labeled with radioactive phosphate by conversion into the glucose-6-P-independent form followed by incubation with [y-azP]ATP. The amount of radioactivity incorporated corresponds to 1.4 phosphate groups per subunit. Incubation with a muscle protein phosphatase led to a liberation of radioactivity and a parallel increase in glucose-6-P-independent synthetase activity. Lineweaver-Burk plots with the purified enzyme, using either UDP-glucose or glycogen as the variable substrate, showed a biphasic curve, as previously observed with cruder enzymes. Increasing concentrations of glucose-6-P led to a gradual elimination of the line corresponding to a higher K, value.