Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica

Abstract

BackgroundThe wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65–70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle.ResultsWood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to ~1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg·ATP and Mg·ADP and inhibited by Mg·GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5°C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing.ConclusionMaintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

“…Another amino acid replacement polymorphism was located on the AMP deaminase gene, which catalyses the deamination of AMP to IMP in skeletal muscle and plays an important role in the regulation of cell energetics. AMP deaminase has been proposed to be crucial for cold adaptation both in vertebrate and in invertebrate species (Dieni & Storey ; Marotta et al . ).…”

“…Another amino acid replacement polymorphism was located on the AMP deaminase gene, which catalyses the deamination of AMP to IMP in skeletal muscle and plays an important role in the regulation of cell energetics. AMP deaminase has been proposed to be crucial for cold adaptation both in vertebrate and in invertebrate species (Dieni & Storey 2008;Marotta et al 2009). In the case of AMP deaminase, outlier SNP status was consistently confirmed within both basins and supported in the large-scale geographical analysis, albeit only with the Bayesian method.…”

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

“…This level of glucose was chosen as it represents a high concentration of cryoprotectant that can potentially be encountered within core organs and blood cells of frozen frogs, and has been in standard use for assessing the effect of high glucose on enzyme kinetic parameters in previous studies (Dieni and Storey, 2008;Dieni and Storey, 2010;Dieni and Storey, 2011). The addition of glucose significantly changed both V max and K m peptide values for skeletal muscle GSK-3 from both control and frozen frogs.…”

“…In particular an 'override' control on normal glucose homeostasis would need to be present to prevent the activation of glycogenesis that is normally triggered when glucose rises above about 10mmoll -1 . Post-translational modification of enzymes is a widespread mode of biochemical regulation in wood frog freeze tolerance, and recent studies have uncovered roles for reversible protein phosphorylation in controlling: (i) glycolysis (Dieni and Storey, 2011); (ii) antioxidant defence and reductive biosynthesis (Dieni and Storey, 2010); (iii) fuel storage and mobilization (Rider et al, 2006;Dieni and Storey, 2009); (iv) adenylate metabolism (Dieni and Storey, 2008);and (v) protein degradation (Woods and Storey, 2006). In the current study we investigated GSK-3 and its regulation by post-translational modification in response to freezing.…”

Regulation of glutamate dehydrogenase and lactate dehydrogenase in the freeze tolerant wood frog, Rana sylvatica

“…Hence, it is not surprising that RPP has an integral role in wood frog freeze tolerance. During freezing events, phosphorylation modulates enzymes of glycolysis enabling the production of cryoprotective agents such as glucose for freezing survival (Dieni and Storey, 2008;Dieni and Storey, 2011). Antioxidant defense enzymes in wood frogs such as catalase (Dawson and Storey, 2016) and superoxide dismutase (Dawson et al, 2015) are also modulated by RPP regulation.…”