PURIFICATION AND CHARACTERIZATION OF GLYCOGEN PHOSPHORYLASE-A AND PHOSPHORYLASE-B FROM THE FREEZE-AVOIDING GALL MOTH LARVAE EPIBLEMA-SCUDDERIANA

The active a and inactive, b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE+ ion exchange and 3'-5'-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3 +/- 0.74 and 2.7 +/- 0.87 mumol glucose-1-P.min-1.g wet weight-1, respectively, in -4-degrees-C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 8 2 units.mg protein-1, respectively. Both enzymes were dimers with native molecular weights of 215000 +/- 18000 for glycogen phosphorylase a and 209000 +/- 15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000 +/- 2000. Both enzymes showed pH optima of 7.5 at 22-degrees-C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10-degrees-C. Michaelis constant values for glycogen at 22-degrees-C were 0.12 +/- 0.004 mg.ml for glycogen phosphorylase a and 0.87 +/- 0.034 mg.ml for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5 +/- 0.07 mmol.l-1 for glycogen phosphorylase a and 23.6 mmol.l-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K(a) of 0.176 +/- 0.004 mmol.l-1. Michaelis constant and K(a) values decreased by two- to fivefold at 5-degrees-C compared with 22-degrees-C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol.l-1) but was inhibitory to both enzyme forms at high concentrations (2 mol.l-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.