Regulation of liver lactate dehydrogenase by reversible phosphorylation in response to anoxia in a freshwater turtle
Lactate dehydrogenase (LDH) is the terminal enzyme of anaerobic glycolysis and key to hypoxia/anoxia survival by most animals. In this study, the effects of anoxic submergence (20h at 7°C in nitrogen-bubbled water) were assessed on LDH from liver of an anoxia-tolerant freshwater turtle, the red-eared slider (Trachemys scripta elegans). Liver LDH from aerobic and anoxic turtles was purified to homogeneity in two steps. The kinetic properties and thermal stability of purified LDH were analyzed, revealing significant differences between the two enzyme forms in Vmax, Km pyruvate, and I50 pyruvate as well as melting temperature determined by differential scanning fluorimetry. The phosphorylation state of aerobic and anoxic forms of LDH was visualized by ProQ Diamond phosphoprotein staining, the results indicating that the anoxic form had a higher phosphorylation state. Incubation studies that promoted protein kinase versus protein phosphatase actions showed that changes in the phosphorylation state of aerobic and anoxic forms mimicked the anoxia-responsive changes in Km pyruvate and I50 pyruvate. The high phosphate form of liver LDH that occurs in anoxic turtles appears to be a less active form. Turtle liver LDH was also subject to another form of posttranslational modification, protein acetylation, with a 70% higher content of acetylated lysine residues on anoxic versus aerobic LDH. This is the first study to show that LDH function in an anoxia-tolerant animal can be differentially modified between aerobic and anoxic states via the mechanism of posttranslational modification.
|Keywords||Acetylation, Anaerobiosis, Enzyme regulation, Liver glycolysis, Phosphorylation|
|Journal||Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology|
Xiong, Z.J. (Zi Jian), & Storey, K. (2012). Regulation of liver lactate dehydrogenase by reversible phosphorylation in response to anoxia in a freshwater turtle. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 163(2), 221–228. doi:10.1016/j.cbpb.2012.06.001