Purification and characterization of aldolase from the cold hardy insect Epiblema scudderiana: Enzyme role in glycerol biosynthesis

Abstract

Aldolase was purified to homogeneity from larvae of the freeze avoiding 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. Aldolase in both 15°C and −4°C acclimated larvae occured in a single enzyme form with an isoelectric point of 5.0–5.1. The pH optimum of the enzyme was 6.0 when assayed in imidazole buffer at 22°C and increased to 6.8 at 5°C. The Arrhenius plot was linear between 5 to 40°C with an activation energy of 68.9 ± 2.31 kJmol −1. K m values for fructose 1,6-bisphosphate increased 2-fold when assay temperature was decreased from 22 to 5°C. No allostetic activators of the enzyme were found but α-glycerophosphate, inorganic phosphate, and glycerol were effective inhibitors under the saturating substrate concentrations that exist for aldolase during glycerol biosynthesis. Inhibitory effects of α-glycerophosphate and inorganic phosphate increased when assayed at 5°C but the opposite was true of inhibition by glycerol. Inhibitory controls on aldolase may function to help bring about the cessation of cryoprotectant synthesis as well as maintain the cryoprotectant glycerol pool over the winter months.