Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866 -2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.

Thioredoxin reductase (TR) is a flavoenzyme, containing one selenocysteine (Sec) residue at the penultimate carboxyl-terminus, that catalyzes the NADPH-dependent reduction of oxidized thioredoxin. Sec is encoded by the UGA stop codon in the open reading frame of the mRNA, and the conserved stem-loop structure in 3'-untranslated regions functions as the determinant of Sec incorporation instead of termination of translation. The efficiency of Sec incorporation in Sec-containing enzymes in physiological or selenium (Se)-deficient condition remains unclear. To clarify this, we have developed monoclonal antibodies to human TR, and established a sandwich enzyme-linked immunosorbent assay to determine TR protein content. We observed that the specific activity of cytosolic TR in NCI-H441 cells increased with increasing concentrations of Se in a serum-free medium. The specific activity of TR purified from each cytosol was essentially equal to the calculated specific activity of each cytosolic TR. The Se content of TR increased with increasing concentration of Se in the medium, from 0.32 mol/mol of TR subunit (no SE) to 0.98 mol/mol of TR subunit (500 nM Se), and was directly correlated with the specific activity of TR. When calculated from the cytosolic TR specific activity of human peripheral mononuclear cell, the theoretical efficiency of Sec incorporation in physiological conditions is assumed to be 87%.