Homogeneous glycogen synthetase b from rat liver.

Glycogen synthetase b has been purified about 2,000-fold from a crude extract of rat liver. The purification procedure consisted of the separation of the glycogen pellet by centrifugation, solubilization of the synthetase by phosphorolysis of the glycogen by endogenous phosphorylase, and adsorption and elution from Ca3(P04)2 gel. The final material appears homogeneous on gel electrophoresis. A subunit molecular weight of 85,000 was obtained from electrophoresis in the presence of sodium dodecyl sulfate. The oligomer appears to have a molecular weight of 260,000 as isolated, suggesting that it is a trimer. On storage in solution in the cold, a species twice this size appears. The peak absorbance of the enzyme was at 278 nm with a value of E::,,, at this wave length of 14.5. The spectrum showed no evidence of conjugated chromophores. Determinations of alkali-labile phosphate and reactive sulfhydryl groups gave values of 12.4 and about 6, respectively. Activity decreased linearly to zero with the titration of the first three sulfhydryl groups (or the first twe when glycogen was present), indicating that at least one of the most reactive groups is essential for activity. Incubation of the purified synthetase at 0” led to inactivation which was reversed on rewarming. Glycogen protected against this cold inactivation.