PURIFICATION AND CHARACTERIZATION OF STARCH PHOSPHORYLASE FROM CUSCUTA-REFLEXA FILAMENTS

Starch phosphorylase (EC.2.4.1.1.) from the filaments of Cuscuta reflexa has been purified using (NH4)(2)SO4 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 400000 +/- 5000. Subunit M(r) of the enzyme as determined by SDS polyacrylamide gel electrophoresis is 100000 +/- 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 degrees, the energy of activation being 12.2 kcal (degrees mol)(-1) and energy of activation for denaturation is 32.94 kcal (degrees mol)(-1). Maximum activity occurred at pH 5.7 with half-maximum activity at pH 5.2 and 7.8.