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

Abstract

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.

Mitochondrial physiology in the skeletal and cardiac muscles is altered in torrent ducks, Merganetta armata, from high altitudes in the Andes

“…The high-altitude population had a high activity of LDH in the heart (Table 1; Fig. 3), similar to what has been observed in various tissues of species that experience cold and hypoxic/anoxic environments (Rosser and Hochachka, 1993;Dawson et al, 2013;Shahriari et al, 2013;Katzenback et al, 2014). However, LDH in the heart likely facilitates lactate oxidation rather than production, a process that our data suggests might be enhanced in highland torrent ducks (Brooks, 1998;Gladden, 2004;Brooks, 2009).…”

Regulation of skeletal Muscle Glycolysis During Dehydration in the Aestivating African Clawed Frog, Xenopus Laevis

“…It has been previously demonstrated that LDH from the liver of X. laevis maintains the control level of activity in vitro when urea is present. Katzenback at al. (2014) concluded that the PTM changes allowed the enzyme to function normally in the face of this protein denaturant.…”

“…In order to survive as long as possible, frogs are thought to reduce their metabolic rate and, when dehydration begins to affect oxygen distribution to tissues, they recruit anaerobic glycolysis to meet ATP demands (Malik and Storey 2009). It has further been demonstrated that X. laevis liver enzymes are modified in response to dehydration in order to adjust their function to the new metabolic demand and to ensure that when urea levels are high the enzymes retain their functionality (Katzenback et al 2014).…”

“…It was demonstrated by Katzenback et al, (2014) that in the presence of physiological concentrations of urea, all the substrate Km values of liver LDH from dehydrated X. laevis were restored to control values. This allows the enzyme to continue functioning normally as urea levels rose over the course of dehydration.…”

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

“…That is, a greater percentage of arterial oxygen was being delivered to tissues in the aestivating frog than control. This relative decrease in oxygen content of venous blood in aestivating X. laevis is likely attributable to lactate-induced plasma acidification caused by enhanced lactate dehydrogenase (LDH) activity in dehydrated frogs at physiological concentrations of urea (Katzenback et al, 2014). At a low pH, hemoglobin has a higher release rate of oxygen and this could allow greater oxygen unloading in peripheral tissues when wholebody water content is reduced (Jokumsen and Weber, 1980).…”