Maintenance of intracellular levels of glutathione (GSH), glutathione disulfide (GSSG) and protein:glutathione mixed disulfides (PrSSG) as well as the relative GSH/GSSG is required for normal cell function. The interaction between both high and low molecular weight monothiols or dithiols and disulfides are normally too slow at physiological pH to take place to any significant extent. A group of enzymes that catalyze these exchange reactions have been identified and are known as thioltransferase (glutaredoxin) (TT,GRX), thioredoxin (TRX) and protein disulfide isomerase (PDI). It is also essential for eukaryotic cells to keep vitamin C and E, known for their antioxidant properties, in their respective reduced forms. Recently, we discovered that TT,GRX and PDI but not TRX nor TRX reductase have physiologically significant dehydroascorbate (DHA) reductase activity. Accordingly, it is proposed that DHA is reduced by GSH, in vivo, catalyzed by TT,GRX or PDI. The purpose of this proposal is to investigate the structure, molecular regulation, and function of the two known mammalian dehydroascorbate reductases. Specifically, it is proposed; 1) to further characterize mammalian TT,GRX and PDI using kinetic, molecular biological, and X-ray crystallographic approaches. 2) To test the hypothesis that dehydroascorbic acid is the immediate oxidant in protein disulfide formation 3) to examine the DHA reductase activity of PDI in relation to its role as the beta-subunit of prolyl 4-hydroxylase, and as an explanation for the synergistic action of GSH and ascorbic acid in collagen formation. 4) to investigate human erythrocytes and granulocytes for the participation of mammalian dehydroascorbate reductases in normal cells. 5) to investigate the role of high levels of TT,GRX in the stomach and small intestine relative to its DHA reductase activity. The TT,GRX and PDI systems may be important links between glutathione metabolism and vitamin C. These studies should open up new approaches to our understanding of the role of dehydroascorbate reductases in several biomedical areas such as cardiovascular disease (myocardial ischemia and reoxygenation damage), drug intoxication, chemical carcinogenesis, diabetes and metabolic defects in collagen, carnitine and catecholamine biosynthesis.
