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 μmol glucose-1-P·min-1·g wet weight-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 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°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml-1 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 Ka of 0.176±0.004 mmol·l-1. Michaelis constant and Ka values decreased by two- to fivefold at 5°C compared with 22°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.
Cryoprotectant synthesis Insect cold hardiness Glycerol metabolism Regulation of glycogenolysis Gall moth, Epiblema
glycogen phosphorylase a
glycogen phosphorylase b
concentration of inhibitor that reduces enzymes velocity by 50%
concentration of activator that produces half-maximal activation of enzyme activity
Michaelis-Menten substrate affinity constant
sodium dodecyl sulphate polyacrylamide gel electrophoresis