Purification and Subunit Structure of Glycogen-Branching Enzyme from Rabbit Skeletal Muscle

Abstract

1,4-.alpha.-Glucan: 1,4-.alpha.-glucan 6-.alpha.-D-(1,4-.alpha.-D-glucano)transferase (branching enzyme) was purified by ammonium sulfate precipitation, chromatography on DEAE-cellulose, fractionation with poly(ethyleneglycol) 6000, chromatography on DEAE-Sepharose and gel filtration on Sephadex G150. The final specific activity was 3000 U[unit]/mg corresponding to a purification of .apprx. 10,000-fold over the muscle extracts. A yield of 0.6 mg of enzyme was obtained from 4000 g muscle within 8 days, corresponding to an overall yield of 7%. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate, and this technique yielded a subunit MW of 77,000. The apparent MW of the native enzyme determined by gel filtration on Sephadex G150 was 60,000, demonstrating that branching enzyme is a monomeric protein. Only a very small proportion of the branching enzyme activity in muscle extracts (2%) precipitated with the protein-glycogen complex. This finding, and its low concentration in muscle, explain why a protein-staining band corresponding to branching enzyme cannot be detected by PAGE of the protein-glycogen complex.