Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog
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Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K m and V max) of G6PDH showed a significant increase in K m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K m NADP+ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K m G6P of 94.1 ± 1.1 μM and K m NADP+ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K m G6P was 172 ± 4.3 μM, K m NADP+ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.
KeywordsEnzyme Wood frog Freeze-tolerance Phosphorylation G6PDH
We thank J.M. Storey for critical commentary on the manuscript. This work was supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs program.
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