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 sulphate 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/mg corresponding to a purification of approximately 10000-fold over the muscle extracts. 0.6 mg of enzyme was isolated from 4000 g muscle within eight days corresponding to an overall yield of 7%. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, and this technique yielded a molecular weight of 77000 for the subunit molecular weight of branching enzyme. The apparent molecular weight of the native enzyme determined by gel filtration on Sephadex G150 was 60000, 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 polyacrylamide gel electrophoresis of the protein-glycogen complex.