Purification and characterization of starch phosphorylase from Cuscuta reflexa filaments

Abstract

Starch phosphorylase (EC.2.4.1.1.) from the filaments of Cuscuta reflexa has been purified using (NH 4) 2SO 4 fractionation, ion-exchange chromatography using DEAE cellulose and gel filtration through Sephadex G-200. The enzyme preparation is estimated to be about 85–90% pure as revealed by native polyacrylamide gel electrophoresis. The native M r of the enzyme as determined by gel filtration through Sephadex G-200 is 400 000 ± 5000. Subunit M r of the enzyme as determined by SDS polyacrylamide gel electrophoresis is 100 000 ± 3000. Unlike other starch phosphorylases, the present enzyme is not inhibited by aromatic amino acids. The kinetic data are characteristic of a sequential reaction mechanism. The optimum temperature for starch phosphorylase enzyme activity is 45°, the energy of activation being 12.2 kcal (°mol) −1 and energy of activation for denaturation is 32.94 kcal (°mol) −1. Maximum activity occurred at pH 5.7 with half-maximum activity at pH 5.2 and 7.8.