Passive regeneration of glutathione: Glutathione reductase regulation from the freeze-tolerant North American wood frog, Rana sylvatica

Abstract

Wood frogs inhabit a broad range across North America, extending from the southern tip of the Appalachian Mountains to the northern boreal forest. Remarkably, they can survive the winter in a frozen state, where as much as 70% of their body water is converted into ice. Whilst in the frozen state, their hearts cease to pump blood, causing their cells to experience ischemia, which can dramatically increase the production of reactive oxygen species within the cell. To overcome this, wood frogs have elevated levels of glutathione, a primary antioxidant. We examined the regulation of glutathione reductase, the enzyme involved in recycling glutathione, in both the frozen and unfrozen (control) state. Glutathione reductase activity from both the control and frozen state showed a dramatic reduction in substrate specificity () for oxidized glutathione (50%) when measured in the presence of glucose (300 mmol l) and a increase (157%) when measured in the presence of levels of urea (75 mmol l) encountered in the frozen state. However, when we tested the synergistic effect of urea and glucose simultaneously, we observed a substantial reduction in the for oxidized glutathione (43%) to a value similar to that with glucose alone. In fact, we found no observable differences in the kinetic and structural properties of glutathione reductase between the two states. Therefore, a significant increase in the affinity for oxidized glutathione in the presence of endogenous levels of glucose suggests that increased glutathione recycling may occur as a result of passive regulation of glutathione reductase by rising levels of glucose during freezing.

Regulation of glutathione-based antioxidant defenses in response to dehydration stress in the African clawed frog, Xenopus laevis

“…Using Expasy, a bioinformatic resource portal that calculates protein molecular weight based on amino acid sequence, the expected subunit molecular weight of X. laevis GR (obtained from the NCBI database (NP_001089322.2)) was ~51.7 kDa. This calculated molecular weight corresponds well with the previous studies of vertebrate amphibians including ~55.1 kDa in Trachemys scripta elegans and ~50 kDa in R. sylvatica (Willmore and Storey, 2007;Dawson and Storey, 2017).…”

“…The results suggest that the frog may be benefitting from a preexisting hyperuremic response to augment the function of GR and promote the recycling of GSSG to GSH for antioxidant defense. A similar result was observed for R. sylvatica muscle GR, with K m GSSG remaining constant between control and frozen conditions but decreasing significantly when exposed to the high glucose levels (used as a cryoprotectant) of the frozen wood frog (Dawson and Storey, 2017).…”

“…Another model organism that must endure elevated ROS generated under ischemic conditions during dormancy is the freeze-tolerant wood frog, Rana sylvatica. GR in the muscle of R. sylvatica showed an increased affinity for GSSG when exposed to physiological glucose concentrations found in a frozen frog (Dawson and Storey, 2017).…”

“…This result demonstrates an environmental stimulation of the enzyme, enhancing substrate affinity during freezing. Therefore, GR activity may be vital to maintain the GSH:GSSG ratio and limit ROS-mediated tissue damage during dormancy in the freezetolerant wood frog (Dawson and Storey, 2017). A similar result could occur in the African clawed frog which is known to accumulate urea as a mechanism of delaying transcutaneous water loss.…”

“…It further investigates whether exposure to urea levels similar to those in a dehydrated frog affects GR enzymatic properties (e.g. substrate affinity) as observed in the muscle of R. sylvatica (Dawson and Storey, 2017). The mechanism(s) by which the enzyme alters its properties under dehydration stress are also investigated by analyzing differences in post-translational modifications, with a focus on phosphorylation-specific changes.…”