PURIFICATION AND PROPERTIES OF GLYCOGEN-PHOSPHORYLASE FROM THE FAT-BODY OF LARVAL MANDUCA-SEXTA

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 K-m 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 Mg2+-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.