Glucose-6-phosphate dehydrogenase is posttranslationally regulated in the larvae of the freeze-tolerant gall fly, Eurosta solidaginis, in response to freezing

The freeze-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose-6-phosphate dehydrogenase (G6PDH) mediates the rate-limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5 degrees C) and frozen (-15 degrees C) E. solidaginis larvae by a single-step chromatography method utilizing 2 ',5 '-ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in K-m for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.