Enzyme activity profiles in an overwintering population of freeze-avoiding gall moth larvae, Epiblema scudderiana

The maximal activities of enzymes of intermediary metabolism, notably glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis were measured over a winter season in freeze-avoiding larvae of the gall moth Epiblema scudderiana. Dynamic changes in enzyme activities were found to reflect metabolic events associated with different and changing requirements of the larvae for survival as winter progressed and ended. Activities of enzymes associated with the cryoprotectant glycerol indicated two possible pathways for its synthesis: (1) glyceraldehyde-phosphate → glyceraldehyde → glycerol via glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or (2) dihydroxyacetonephosphate → glycerol-3-phosphate → glycerol via glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Glycogen phosphorylase activation in the fall supplied carbon equivalents required for glycerol synthesis from glycogen. Hexose monophosphate shunt enzyme activity was high, reflecting the role of this pathway in supplying NAD(P)H for glycerol synthesis. Spring clearance of glycerol appeared to occur via polyol dehydrogenase and glyceraldehyde kinase. Increasing fructose-bisphosphatase activity into late winter and spring was found to increase the gluconeogenic potential needed for cryoprotectant removal. Increased activity of glycerol-3-phosphate dehydrogenase in the spring, possibly reflecting increased α-glycero-phosphate shuttle activity, may be key to the removal of reducing equivalents generated from glycerol removal.

Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect

Phosphofructokinase (PFK) from larvae of the freeze-avoiding gall moth Epiblema scudderiana was purified 711-fold using ATP-agarose affinity chromatography to a final specific activity of 23 U/mg protein. The native molecular mass of the enzyme was 420 000 ± 20 000 Da. The enzyme showed an optimum pH of 8.13 ± 0.21 at22 °Cand 8.19 ± 0.11 at5 °C. Arrhenius plots of PFK activity showed a sharp break at 9 °C. S0.5 values for fructose 6-phosphate showed positive thermal modification, decreasing with decreasing assay temperature; the opposite was true for ATP-Mg2+. PFK was activated by fructose 2,6-bisphosphate, AMP, and inorganic phosphate; activator effects were temperature-dependent. The enzyme was inhibited by ATP-Mg2+, citrate-Mg2+, and phosphoenolpyruvate. The positive effects of low temperature on enzyme kinetic properties would promote PFK activity to channel glycolytic carbon flow into the production of glycerol during cold-stimulated cryoprotectant synthesis.

Cryoprotectant production capacity of the freeze-tolerant wood frog, Rana sylvatica

Freezing survival of the wood frog (Rana sylvatica) is enhanced by the synthesis of the cryoprotectant glucose, via liver glycogenolysis. Because the quantity of glucose mobilized during freezing bears significantly on the limit of freeze tolerance, we investigated the relationship between the quantity of liver glycogen and the capacity for cryoprotectant synthesis. We successfully augmented natural levels of liver glycogen by injecting cold-conditioned wood frogs with glucose. Groups of 8 frogs having mean liver glycogen concentrations of 554 ± 57 (SE), 940 ± 57, and 1264 ± 66 μmol/g catabolized 98.7, 83.4, and 52.8%, respectively, of their glycogen reserves during 24 h of freezing to −2.5 °C. Glucose concentrations concomitantly increased, reaching 21 ± 3, 102 ± 23, and 119 ± 14 μmol/g, respectively, in the liver, and 15 ± 3, 42 ± 5, and 61 ± 5 μmol/mL, respectively, in the blood. Because the capacity for cryoprotectant synthesis depends on the amount of liver glycogen, the greatest risk of freezing injury likely occurs during spring, when glycogen reserves are minimal. Non-glucose osmolites were important in the wood frog's cryoprotectant system, especially in frogs having low glycogen levels. Presumably the natural variation in cryoprotectant synthesis capacity among individuals and populations of R. sylvatica chiefly reflects differences in glycogen reserves; however, environmental, physiological, and genetic factors likely are also involved.