Glucose-6-phosphate dehydrogenase in cold hardy insects: Kinetic properties, freezing stabilization, and control of hexose monophosphate shunt activity

Abstract

Properties of glucose-6-phosphate dehydrogenase were assessed from the larvae of three insect species, the freeze tolerant Eurosta solidaginis, the freeze avoiding Epiblema scudderiana, and warm-acclimated Tenebrio molitor. Maximal enzyme activities were 16–17 fold higher in the cold hardy larvae than in T. molitor in line with the key role of G6PDH in providing NADPH for the synthesis of cryoprotectant polyols in these species. K_m values for glucose-6-P and NADP were determined at both high (24°C) and low (4°C) temperatures for all three enzymes. Temperature decrease had the greatest effect on T. molitor G6PDH increasing K_m glucose-6-P by 3-fold and K_m NADP by 2-fold; K_m values for G6PDH from the cold hardy species were less temperature-sensitive. The addition of polyols (glycerol, sorbitol) or KCl caused selected changes in the K_m values for both substrates in all species. Cryoprotectant action in the freezing protection of G6PDH was also examined, comparing G6PDH from E. solidaginis, E. scudderiana and yeast. A range of polyols (glycerol, sorbitol), other carbohydrates (trehalose, glucose, lactate) and amino acids (alanine, glutamate, proline) were effective in protecting activity during freezing. Without cryoprotectant, enzyme activity after 1 h freezing at −77°C was <10% compared to controls. Low concentrations of protectants (typically <50 mM) gave complete protection during freezing. Values for cryoprotectant concentrations giving half-maximal recovery of activity ranged from as low as 7–10 mM for trehalose to 20–25 mM for most other compounds.