| Summary: | The freshwater turtle, Trachemys scripta is considered one of the most anoxia-tolerant vertebrates because of its ability to survive months at cold temperatures in the complete absence of oxygen. When deprived oxygen, mitochondria from anoxia intolerant organisms become one of the largest cellular energy consumers because of the reverse functioning of the F₁F₀-ATPase (complex V), which hydrolyzes ATP to pump protons out of the mitochondrial matrix, quickly depleting cellular ATP and leading to cellular death. T. scripta has previously shown to inhibit complex V in response to anoxia exposure, but the regulatory mechanism is still unknown. The goal of this thesis was to explore the mitochondrial response to anoxia in T. scripta. My first objective was to deduce the mechanism responsible for the severe downregulation of Complex V. In heart, brain, and liver tissue from anoxic exposed turtles, complex V activity was significantly reduced to more than 80% compared with normoxic controls. Employing a proteomics approach, I determined that three subunits of complex V (ATP5A1, ATP5F1, and MT-ATP5J), all associated with the peripheral stalk, decreased in protein expression in response to anoxia. Increasing assay buffer pH, in an attempt to strip Inhibitory Factor-1 (IF₁) from complex V did not increase enzyme activity of normoxic or anoxic exposed samples, but decreasing pH < 7.5 decreased activity and at pH 6.0 there was no significant difference in activity between normoxic-and anoxic-exposed samples. Stimulating endogenous phosphatases slightly reduced activity in normoxic samples but had no effect on anoxic samples. Nitric oxide did not have a significant effect on complex V activity as previously seen in mice. The second objective of this thesis was to characterize the changes in proteins involved in mitochondrial function during anoxia. Proteomic analysis revealed differential expression of numerous enzymes involved with the electron transport system (ETS), the tricarboxyl acid (TCA) cycle, as well as lipid and amino acid metabolism. Overall, results from this thesis demonstrate that mitochondria from turtles alter protein expression of numerous proteins in response to anoxia and that reduced complex V activity is likely related to decreases in the expression of specific complex V subunits. === Science, Faculty of === Zoology, Department of === Graduate
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