Reversible phosphorylation regulation of NADPH-linked polyol dehydrogenase in the freeze-avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism.

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH-dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K(m) for glyceraldehyde in 5°C-acclimated larvae was 7.0 mM and doubled in - 15°C-exposed larvae. This change suggested that PDH is regulated by a state-dependent covalent modification. Indeed, high and low K(m) forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K(m) glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K(m) of the - 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from - 15°C-acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the - 15°C enzyme. These experiments established that PDH is regulated by state-dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months.