This proposal is aimed at elucidating the physiological role of thioredoxins. Thioredoxins are low molecular weight (about 12 Kd) dithiol proteins that have been implicated in a variety of roles involving thio-related reactions. They are ubiquitously distributed, present in high concentrations (about 50 MuM in E. coli), and they have a conserved active site sequence in organisms ranging from bacteria to mammals. Since the true in vivo role of this class of proteins is not certain we will explore several possibilities: 1. Does thioredoxin act as the cell's defense against free radical damage? Bacterial stains constructed to overproduce thioredoxin to 150-fold show protection against Gamma-rays in proportion to thioredoxin content. We plan to test the hypothesis that thioredoxin acts as a radical scavenger in vivo using strains lacking glutathione and in vitro testing the ability of thioredoxin to compete with known radical scavengers. 2. What is the physiological basis for thioredoxin phosphorylation? Thioredoxin is phosphorylated in late log cells and phosphoryation levels change in response to media supplements. We plan to establish what medium components and growth conditions affect phosphorylation levels. In vitro conditions will be defined for phosphorylation to permit biochemical studies to test the hypothesis that thiorecdoxin couples phosphate transfer (i.e. to nucleotide acceptors) to oxidation to the disulfide form. 3. Where and how is thioredoxin localized in the cell? An important clue to thioredoxin's role (in E. coli) may be related to its localization in a region of the cell which is osmotically sensitive yet contained within the inner membrane. The nature of this compartment and its topological relation to the membrane fusion sites of Bayer's junctions will be explored by EM using antibodies raised to the active site sequence; chemical crosslinking will be used to define thioredoxin nearest neighbors. 4. Does thioredoxin modulate the activity of various enzymes by controlling their thiol oxidation state? We plan to assess the oxidation state of thioredoxin under different growth conditions and to attempt isolation of stable mixed disulfide forms of thioredoxin with target proteins.
