A hydrogen peroxide safety valve: The reversible phosphorylation of catalase from the freeze-tolerant North American wood frog, Rana sylvatica

Background: The North American wood frog, Rana sylvatica, endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H2O2 may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H2O2 into molecular oxygen and water, was investigated as a result. Methods: The present study investigated the purification and kinetic profile of CAT in relation to the phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica. Results: Catalase from skeletal muscle of frozen wood frogs showed a significantly higher V-max (1.48 fold) and significantly lower K-m for H2O2 (0.64 fold) in comparison to CAT from control frogs (5 degrees C acclimated). CAT from frozen frogs also showed higher overall phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (127 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the K-m for H2O2 of CAT, whereas protein phosphatase 2B or 2C action significantly increased the K-m. Conclusion: The physiological consequence of freeze-induced CAT phosphorylation appears to improve CAT function to alleviate H2O2 build-up in freezing frogs. General significance: Augmented CAT activity via reversible phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia. (C) 2015 Elsevier B.V. All rights reserved.