Regulation of hexokinase in a freeze avoiding insect: Role in the winter production of glycerol

Hexokinase from larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana, was purified 20-fold using chromatography on DE52 Sephadex, p hosphocellulose, and blue dextran. Final specific activity was 75.8 U/mg an d SDS-PAGE gave a molecular weight of 94,000 for the monomer. Arrhenius plo t showed a break at 16 degrees or 12 degreesC in the absence vs. presence o f 10% v/v glycerol, indicating a conformational change in the enzyme at low er temperatures but suggesting a stabilizing effect of glycerol. Comparison of hexokinase kinetic properties at 22 degrees and 4 degreesC showed highe r affinity for both glucose and ATP (Km values were 45-50% lower), as well as for the cofactor Mg2+, at the lower temperature. Furthermore, product in hibition by glucose-6-phosphate and ADP was reduced at 4 degreesC. Glucose levels rise in E. scudderiana as an apparent by-product of high rates of gl ycogenolysis during glycerol synthesis. The temperature-dependent propertie s of hexokinase would facilitate the recycling of this glucose back into th e pathway of glycerol synthesis and could help to achieve the near stoichio metric conversion of glycogen to glycerol that is seen during cold hardenin g. (C) 2001 Wiley-Liss, Inc.