PubMed1975
Essentially glycogen-free, fully converted rabbit muscle glycogen synthase I and D forms were purified to a specific activity of 30 approximately 35 units/mg, higher than that previously reported. Polyacrylamide gel electrophoresis of the synthase I and D forms in the presence of sodium dodecyl sulfate revealed two species with molecular weights of 85,000 and 81,000 (I form) and one species with molecular weight of 85,000 (D form), respectively. The 81,000 subunit of the synthase I form is a product of proteolysis, since its proportion decreased when the enzyme was purified in the presence of a proteinase inhibitor and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Two-dimensional chromatography and high voltage electrophoretic maps of the peptides produced by exhaustive tryptic digestion of synthase I and D forms gave 78 and 85 peptides, respectively. These values agreed with the expected theoretical number of peptides assuming that the molecular weight of the enzyme subunit is 85,000 based on the analytical data of arginine and lysine content. Pro-Leu- was found as the NH2-terminal dipeptide sequence of synthase D form by the dansyl Edman method. The automated sequential degradation of synthase I form by the method of Edman provided the following NH2-terminal octapeptide sequence: Pro-Leu-Ser-Ser-Thr-Leu-Ser-Val-. The molecular weight of the subunit was also determined to be 80,000 approximately 90,000 by the quantitative analysis of phenylthiohydantoinproline. The purified synthase I and D forms each show two protein bands on gel electrophoresis in the absence of sodium dodecyl sulfate, which were also found to be enzymatically active by employing an activity stain directly on the gels. The molecular species of the I and D forms have molecular weights of 155,000 and 340,000 (I form), and 278,000 and 350,000 (D form), respectively, as measured by gel electrophoresis. Thus, the native synthase I and D forms as visualized on gels under these conditions consist of dimer and tetramer, and trimer and tetramer, respectively.