Glycogen degradation by alpha-glucosidase from a hibernating ground squirrel, Spermophilus lateralis, and a freeze tolerant frog, Rana sylvatica
Glycogen degradation in cells can proceed via two routes, the well-known phosphorolytic pathway via glycogen phosphorylase (GP) and the glucosidic pathway involving alpha-glucosidase. The present study analyzes alpha-glucosidase and its potential for net contributions to organ glucose production in two species that exhibit unusual glucose requirements while overwintering at low body temperatures: the hibernating golden-mantled ground squirrel, Spermophilus lateralis, and the freeze-tolerant wood frog, Rana sylvatica. Mammalian liver (ground squirrel, rat) possessed two neutral isozymes of alpha-glucosidase and one acidic form (pH optima 7.0 and 4.0) whereas amphibian liver (wood frog, leopard frog) had one neutral and one acidic enzyme. Tissue distribution of acidic and neutral forms revealed highest activities in liver and kidney and significant changes in activity during hibernation or freezing in selected tissues. Compared with the amount of active GP, acid alpha-glucosidase could make a substantial contribution to net glucose production in several wood frog organs, notably heart. Liver isozymes were separated by isoelectrofocusing prior to characterization of substrate specificity and affinity, inhibitor effects, and thermal denaturation. Neutral isozymes of all species were similar showing limited use of glycogen, strong thermal denaturation at 45°C and above, and generally reduced substrate affinity at the low temperatures characteristic of the hibernating state. Acidic isozymes used glycogen, showed good thermal stability and substrate affinities were largely temperature-independent. Inhibition of acidic alpha-glucosidase by salts and carbohydrates was reduced for the wood frog enzyme, compared with the leopard frog, a factor that may improve enzyme function in the frozen state.