Clark Holden, M.Sc. Biology 1991

Purification and characterization of glycogen phosphorylase A and 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 micromol glucose-1-P/min/g wet weight, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units/mg protein, respectively. Both enzymes were dimers with native molecular weights of 2l5 000 + l8 000 for glycogen phosphorylase a and 209 000+15 000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87 000+2 000. Both enzymes showed pH optima of 7.5 at 22 °C and a break in the Arrhenius relationship with a two- to fourfold increase in activation energy below 10 °C. Michaelis constant values for glycogen at 22 °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 for glycogen phosphorylase a and 23.6 mmol/L for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a Ka of 0.176+0.004 mmol/L. 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) but was inhibitory to both enzyme forms at high concentrations (2 mol/L). 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 glyeogen phosphorylase b.