Background: Freshwater turtles, Trachemys scripta elegans, are one of the few vertebrate species capable of surviving prolonged periods without oxygen. Anoxic survival requires numerous physiological and biochemical changes, including a drastic reduction in metabolic rate, a cessation of oxygen-based metabolism, and a suppression of urea-synthesis. Given this state, the purpose of this study was to investigate the possible regulation of liver glutamate dehydrogenase (GDH), a key enzyme in both nitrogen and carbohydrate metabolism, when these freshwater turtles transition from normoxic to anoxic conditions.
Methods: GDH was purified to electrophoretic homogeneity using a combination of blue-agarose and GTP-agarose chromatography. Subsequent kinetic analysis of GDH derived from the liver of both control (normoxic) and 20 h anoxic turtles was performed spectrophotometrically. ProQ Diamond phosphoprotein staining was used to determine if GDH was present in differently phosphorylated forms between normoxic and anoxic states, and in vitro incubations with alkaline and acid phosphatases were used to determine if changes in phosphorylation state resulted in kinetic changes.
Results: Kinetic studies revealed that the anoxic form of GDH was significantly less active than the aerobic control, as well as more susceptible to pH-induced inactivation, and GTP inhibition. ProQ Diamond phosphoprotein staining indicated that anoxic liver GDH was significantly less phosphorylated than control GDH. Subsequent stimulation of exogenous alkaline and acid phosphatase activity significantly lowered the Km α-ketoglutarate for control GDH to a value similar to the value found for anoxic GDH in its native state.
Conclusion: We conclude that effector molecules, tissue acidification and reversible phosphorylation act in concert to suppress GDH during anoxia in accordance with general metabolic rate depression and the overall shutdown of oxygen-based metabolism.