REVERSIBLE PHOSPHORYLATIONREGULATION OF NADPH-LINKEDPOLYOL DEHYDROGENASE IN THEFREEZE-AVOIDING GALL MOTH,Epiblema scudderiana:ROLEIN GLYCEROL METABOLISM

摘要

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freezeavoidance strategy of cold hardiness to survive the winter. A key metabolicadaption that supports subzero survival is the accumulation of largeamounts of glycerol as a colligative antifreeze. Production of glycerolrelies on polyol dehydrogenase (PDH) which catalyzes the NADPHdependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties asa result of subzero temperature acclimation; the K_m for glyceraldehyde in5 deg C-acclimated larvae was 7.0mM and doubled in -15 deg C-exposedlarvae. This change suggested that PDH is regulated by a statedependent covalent modification. Indeed, high and low Km forms could be interconverted by incubating larval extracts in vitro under conditionsthat stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K_m glyceraldehyde of the 5 deg C enzyme, whereas protein phosphatase incubations decreased the Kmof the -15 deg C enzyme by about 50%. PDH was purified by ion exchangeand affinity chromatography steps and then subjected to electrophoresis.Staining with ProQ Diamond phosphoprotein stain showed a muchhigher phosphate content of PDH from -15 deg C-acclimated larvae, aresult that was further confirmed by immunoblotting that showed a muchgreater phosphoserine content on the -15 deg C enzyme. These experiments established that PDH is regulated by state-dependent reversible phosphorylation in E. scudderiana and suggest that this regulatorymechanism makes a significant contribution to controlling the synthesis,maintenance, and degradation of glycerol pools over the winter months.