Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis

The African clawed frog, Xenopus laevis, is able to withstand extremely arid conditions by estivating, in conjunction with dehydration tolerance and urea accumulation. Estivating X. laevis reduce their metabolic rate and recruit anaerobic glycolysis, driven by lactate dehydrogenase (LDH; E.C. 1.1.1.27) enzymes that reversibly convert pyruvate and NADH to lactate and NAD+, to meet newly established ATP demands. The present study investigated purified LDH from the liver of dehydrated and control X. laevis. LDH from dehydrated liver showed a significantly higher K m for l-lactate (1.74 fold), NAD+ (2.41 fold), and pyruvate (1.78 fold) in comparison to LDH from the liver of control frogs. In the presence of physiological levels of urea found in dehydrated animals, the K m values obtained for dehydrated LDH all returned to control LDH K m values. Dot blot analysis showed post-translational modifications may be responsible for the kinetic modification as the dehydrated form of LDH showed more phosphorylated serine residues (1.54 fold), less methylated lysine residues (0.43 fold), and a higher level of ubiquitination (1.90 fold) in comparison to control LDH. The physiological consequence of dehydration-induced LDH modification appears to adjust LDH function in conjunction with urea levels in dehydrated frogs. When urea levels are high during dehydration, LDH retains its normal function. Yet, as urea levels drop during rehydration, LDH function is reduced, possibly shunting pyruvate to the TCA cycle

Free-radical first responders: The characterization of CuZnSOD and MnSOD regulation during freezing of the freeze-tolerant North American wood frog, Rana sylvatica

Background: The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O2-•) to molecular oxygen and hydrogen peroxide.Methods: The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica.Results: CuZnSOD from frozen muscle showed a significantly higher Vmax (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O2-• (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (Cm) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different.Conclusion: The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs.General significance: Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia

Cytokine and Antioxidant Regulation in the Intestine of the Gray Mouse Lemur (Microcebus murinus) During Torpor

During food shortages, the gray mouse lemur (Microcebus murinus) of Madagascar experiences daily torpor thereby reducing energy expenditures. The present study aimed to understand the impacts of torpor on the immune system and antioxidant response in the gut of these animals. This interaction may be of critical importance given the trade-off between the energetically costly immune response and the need to defend against pathogen entry during hypometabolism. The protein levels of cytokines and antioxidants were measured in the small intestine (duodenum, jejunum, and ileum) and large intestine of aroused and torpid lemurs. While there was a significant decrease of some pro-inflammatory cytokines (IL-6 and TNF-α) in the duodenum and jejunum during torpor as compared to aroused animals, there was no change in anti-inflammatory cytokines. We observed decreased levels of cytokines (IL-12p70 and M-CSF), and several chemokines (MCP-1 and MIP-2) but an increase in MIP-1α in the jejunum of the torpid animals. In addition, we evaluated antioxidant response by examining the protein levels of antioxidant enzymes and total antioxidant capacity provided by metabolites such as glutathione (and others). Our results indicated that levels of antioxidant enzymes did not change between torpor and aroused states, although antioxidant capacity was significantly higher in the ileum during torpor. These data suggest a suppression of the immune response, likely as an energy conservation measure, and a limited role of antioxidant defenses in supporting torpor in lemur intestine