Efficiency of selenocysteine incorporation in human thioredoxin reductase.
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%.
Yarimizu
J
J
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Nakamura
H
H
Yodoi
J
J
Takahashi
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D003062:Codon / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003062:Codon; D012333:RNA, Messenger; D017279:Selenocysteine; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium