Front line antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica: An in-depth analysis of mechanisms of enzyme regulation
The wood frog, Rana sylvatica, is one of few species that can survive whole-body freezing during overwintering. Frogs endure freezing of up to 70% of their total body water, and demonstrate a complete lack of respiration, heart beat and brain activity. Freezing imposes multiple stresses including anoxia/ischemia, cellular dehydration when water is lost to extracellular ice masses, wide temperature changes, and potential physical damage by ice. One crucial adaptation for freezing survival is well-developed antioxidant defenses to protect tissues from abiotic stress while frozen and deal with rapid changes in the generation of reactive oxygen species associated with anoxia and reoxygenation over freeze/thaw cycles.
This thesis explores the properties and regulation of key antioxidant enzymes, purified via novel schemes, from frog muscle – both Cu/Zn- and Mn-dependent isoforms of superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT). The studies show that changes in activity, stability, and substrate affinity of antioxidant enzymes during the frozen state may be significant preparatory mechanisms employed by R. sylvatica to support the transition from frozen to thawed states and deal effectively with oxidative stress accompanying reperfusion. Moreover, reversible protein phosphorylation plays a central role in regulating the activity of these enzymes to suit physiological needs throughout freeze-thaw cycles. For example, CuZnSOD from muscle of frozen frogs showed a significantly higher Vmax compared to the control enzyme. Muscle MnSOD from frozen frogs showed a significantly lower Km for O2-, higher phosphorylation, and increased enzyme stability compared to control MnSOD. GR from frog muscle showed a significantly lower Km for GSSG in the face of physiological levels of glucose encountered during freezing, as well as the potential for phosphorylation via endogenous kinases. CAT from muscle of frozen frogs showed a significantly lower Km for H2O2 and a higher level of phosphorylation; furthermore, stimulation of endogenous kinases decreased Km H2O2 similar to what occurred in muscle of frozen animals. This thesis provides compelling evidence for regulation of antioxidant enzymes via reversible protein phosphorylation and augmentation of key antioxidant enzymes during freezing of the frog, likely in preparation to endure oxidative stress encountered during reperfusion over winter freeze-thaw cycles.