PURIFICATION AND CHARACTERIZATION OF ALDOLASE FROM THE COLD HARDY INSECT EPIBLEMA-SCUDDERIANA - ENZYME ROLE IN GLYCEROL BIOSYNTHESIS

Aldolase was purified to homogeneity from larvae of the freeze avoidin g gall moth, Epiblema scudderiana to a final specific activity 16.5 U/ mg protein. The tetrameric enzyme had a native molecular weight of 160 +/- 11 kDa and a subunit molecular weight of 37.8 +/- 1.0 kDa. Aldola se in both 15 degrees C and -4 degrees C acclimated larvae occurred in a single enzyme form with an isoelectric point of 5.0-5.1. The pH opt imum of the enzyme was 6.0 when assayed in imidazole buffer at 22 degr ees C and increased to 6.8 at 5 degrees C. The Arrhenius plot was line ar between 5 and 40 degrees C with an activation energy of 68.9 +/- 2. 31 kJmol(-1). K-m values for fructose 1,6-bisphosphate increased a-fol d when assay temperature was decreased from 22 to 5 degrees C. No allo steric activators of the enzyme were found but alpha-glycerophosphate, inorganic phosphate, and glycerol were effective inhibitors under the saturating substrate concentrations that exist for aldolase during gl ycerol biosynthesis. Inhibitory effects of alpha-glycerophosphate and inorganic phosphate increased when assayed at 5 degrees C but the oppo site was true of inhibition by glycerol. Inhibitory controls on aldola se may function to help bring about the cessation of cryoprotectant sy nthesis as well as maintain the cryoprotectant glycerol pool over the winter months.