Rabbit liver glycogen synthase. Purification and comparison of the properties of glucose-6-P-dependent and glucose-6-P-independent forms of the enzyme.
Methods are described for the purification, close to homogeneity, of rabbit liver glycogen synthase in forms dependent on (D-form) or independent (I-form) of glucose-6-P for activity. In previous studies (Camici, M., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol. Chem. 257, 9898-9901), the D-form enzyme was shown to have apparent subunit molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Mapp) of 90,000 and to be susceptible to partial proteolytic degradation. We report here that the purified I-form consisted of a single polypeptide of Mapp = 85,000, even when protease inhibitors were present during the purification. However, appropriate phosphorylation of the I-form enzyme led to a decrease in the electrophoretic mobility of the subunit to generate a species of Mapp = 90,000, identical to that of the D-form. Exposure of the I-form enzyme (subunit Mapp = 85,000) to trypsin caused degradation in the sequence 85,000----82,000----79,000----72,000; concomitantly, the enzyme underwent partial inactivation whether assayed in the presence or absence of glucose-6-P. As purified, the I-form enzyme had a Vmax, determined from variation of UDP-glucose concentration, some 35 times greater than that of the D-form. The UDP-glucose concentration necessary for half-maximal activity was not greatly different, in the range 1-2 mM, for the two enzyme forms.
D000818:Animals; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D005958:Glucosephosphates; D046911:Macromolecular Substances; D019298:Glucose-6-Phosphate; D006006:Glycogen Synthase; D014357:Trypsin; D014532:Uridine Diphosphate Glucose