Purification and characterization of glycogen phosphorylase from Drosophila melanogaster

Abstract

Glycogen phosphorylase b was purified from Drosophila melanogaster with a yield of 15% by low speed centrifugation, DEAE-Sepharose CL-6B chromatography and affinity chromatography on 5′-AMP-Sepharose 4B. The preparation proved to be homogeneous by SDS-polyacrylamide gel electrophoresis and showed a subunit molecular weight of 95,000. Gel filtration of the native enzyme on Sephacryl S-300 revealed a molecular weight of 176,000 suggesting that phosphorylase b is a dimer composed of two identical subunits. The fruit fly phosphorylase b has a maximal specific activity of 36 U/mg when assayed in the direction of glycogen synthesis. It requires AMP for activity ( A 0.5 = 0.4 mM, Hill coefficient: 1.8). The K M for glycogen is 0.4% and the S 0.5 for glucose-1-phosphate is 20 mM (Hill coefficient: 1.3). The enzyme is inhibited by glucose, UDPG, caffeine, glucose-6-phosphate, ATP and IMP.