The phosphorylated and unphosphorylated forms of bovine corpus luteum phosphorylase were purified by ammonium sulfate fractionation, DEAE-cellulose, and Sephadex G-200 column chromatography. The purified enzyme appeared to be homogeneous as determined by electrophoresis in polyacrylamide gel and antigen-antibody precipitation in agar. Molecular weight determination by sucrose density gradient ultracentrifugation gave a value of 185,000 for both forms of the enzyme. Kinetic experiments in the direction of glycogen synthesis showed substrate activation of the unphosphorylated enzyme by glucose 1-phosphate. This substrate activation was abolished at high (0.4 m) concentrations of sodium sulfate. No substrate activation was observed for the phosphorylated enzyme. The activation at high substrate concentration was attributed to an increase in the dissociation rate of (ES), the enzyme-substrate complex (attributed to a conformational change), to free enzyme + products. The substrate activation phenomenon observed here is consistent with the mechanism of negative cooperativity discussed by Conway and Koshland. Unphosphorylated corpus luteum phosphorylase was reversibly activated by sodium sulfate. Kinetic studies in the presence of sodium sulfate indicate that this salt acts as an allosteric activator. The mechanism of this activation is discussed. The phosphorylated enzyme was inhibited by sodium sulfate and the inhibition was competitive with respect to glucose 1-phosphate. Antibody prepared against phosphorylated corpus luteum phosphorylase gave a reaction of identity against bovine liver phosphorylase, a reaction of partial identity against human leukocyte phosphorylase, but showed no cross reactivity with bovine, rabbit, or human muscle phosphorylase. The similarity in the molecular weights of the phosphorylated and unphosphorylated enzyme, their behavior in the presence of high salt concentration, and the immunological findings indicate that glycogen phosphorylase from corpus luteum and liver are closely related.
ASJC Scopus subject areas