PURIFICATION AND PROPERTIES OF RAT MUSCLE GLYCOGEN PHOSPHORYLASE

The purification and crystallization of rat muscle glycogen phosphorylase (α-1,4-glucanorthophosphate glucosyl transferase, EC 2.4.1.1) are described and its properties compared to those of rabbit muscle phosphorylase. The purified enzyme has a specific activity of 85 ± 5 units per mg, corresponding to a turnover number of 15,700 moles of substrate consumed/mole of enzyme dimer per min when measured at 30° in the direction of glycogen synthesis. Molecular weights of 185,000 and 350,000 were calculated for the b (dimer) and a (tetramer) forms of the enzyme, both by sedimentation velocity and diffusion and by equilibrium measurements. As found for rabbit muscle phosphorylase (Seery, V. L., Fischer, E. H., and Teller, D. C. (1967), Biochemistry 6, 3315) the dimeric form of the enzyme appears to be slightly associated and the tetrameric form slightly dissociated. Amino acid analyses carried out on three separate preparations of rat phosphorylase gave essentially identical results. Comparison with amino acid analyses performed under similar conditions on rabbit phosphorylase indicated a considerable degree of similarity between the two muscle enzymes. Whereas no amino- or carboxyl-terminal group had been detected in any phosphorylase so far investigated, 1 equiv of isoleucine/enzyme monomer was released by carboxypeptidase A attack, without loss of enzymatic activity. Subsequent digestion with carboxypeptidase B released a single lysyl residue, indicating that rat phosphorylase possesses a lysylisoleucine sequence at its carboxyl end. A phosphorylated tetradecapeptide was isolated from the site involved in the phosphorylase b into a conversion. Its amino acid sequence was found to be identical with that of a phosphotetradecapeptide isolated from rabbit muscle phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964), Biochemistry 3, 542), except for the conservative substitution of an aspartyl for a glutamyl residue found in the rabbit phosphopeptide. The above physical and chemical properties together with the finding that the interconversions of the a and b form of rat muscle phosphorylase are catalyzed by rabbit muscle phosphorylase kinase (EC 2.7.1.38) and phosphatase (EC 3.1.3.17) indicate extreme similarity between the two systems. © 1969, American Chemical Society. All rights reserved.