The oxidation of enzyme sulfhydryl groups to disulfides may result in decreased or increased activity (Km or Vmax effects), changes in aggregation state, or changes in protein stability. Whether or not intracellular enzymes undergo changes in oxidation state in vivo (with corresponding changes in function) will depend on the oxidation potential of the protein sulfhydryl group, the kinetics of the oxidation-reduction processes, and the thiol/disulfide status of the cellular environment. The long-term goal of the proposed research is to investigate the relationships among protein thiol oxidation, changes in activity or function, and changes in the intracellular thiol/disulfide status. The thiol/disulfide redox potential relative to the glutathione redox system (Keq for the reaction: Ered + nGSSG yields or reversible action Eox + mGSH) will be measured for rat liver microsomal and freeze-thaw solubilized HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. This particular enzyme has been shown to be extremely sensitive to sulfhydryl group oxidation. The redox potential measurements will be extended to other key enzymes such as acetyl-CoA carboxylase, phosphoenolpyruvate carboxy kinase, fructose-1,6-bisphosphatase, and pyruvate dehydrogenase complex. The effect of oxidation state on net protein stability will also be investigated. Changes in the in vivo thiol/disulfide status in response to dietary manipulation, hormones, and oxidative stress induced by redox active drugs, will be measured by a new technique for the determination of GSH and GSSG in freeze-stopped perfused rat liver preparations. Experiments are proposed which will utilize covalent chromatography in conjunction with immunological detection to determine in vivo changes in the protein sulfhydryl oxidation state of specific proteins in response to changes in the intracellular thiol/disulfide status. Additional in vivo experiments will be performed to determine if the rate of appearance of specific oxidized proteins (if they can be detected) is sufficiently fast to respond to rapid changes in the cellular glutathione status induced by redox active compounds or hormones. These experiments will allow a critical assessment as to whether or not the extent of oxidation of specific intracellular proteins changes in responses to the cellular thiol/disulfide status.
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