title: Purification and properties of glycogen phosphorylase from the fat body of larval Manduca sexta.
abstract: Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.
authors.affiliation: Department of Biochemistry, University of Arizona, Tucson 85721.
authors.forename: E L
authors.initials: EL
authors.lastname: Arrese
authors.forename: B I
authors.initials: BI
authors.lastname: Rojas-Rivas
authors.forename: M A
authors.initials: MA
authors.lastname: Wells
mesh_terms: D000818:Animals; D005216:Fat Body / Q000201:enzymology*; D007814:Larva / Q000201:enzymology; D018402:Manduca / Q000201:enzymology*; D010762:Phosphorylase a / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010763:Phosphorylase b / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits
chemical_list: D010762:Phosphorylase a; D010763:Phosphorylase b