Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells.
Mammalian selenocysteine-containing thioredoxin reductase (TR) isolated from HeLa cells and from human lung adenocarcinoma cells was separated into two major enzyme species by heparin-agarose affinity chromatography. The low-affinity enzyme forms that were not retained on heparin agarose showed strong crossreactivity in immunoblot assays with anti-rat liver TR polyclonal antibodies, whereas the high-affinity enzyme forms that were retained by the heparin column were not detected. Both low and high heparin-affinity enzyme forms contained FAD, were indistinguishable on SDS/PAGE analysis, and exhibited similar catalytic activities in the NADPH-dependent DTNB [5,5'-dithiobis(2-nitrobenzoate)] assay. The C-terminal amino acid sequences of 75Se-labeled tryptic peptides from lung adenocarcinoma low- and high heparin-affinity enzyme forms were identical to the predicted C-terminal sequence of human placental TR. These two determined peptide sequences were -Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Secys-(Gly). Occurrence of the Se-carboxymethyl derivative of radioactive selenocysteine in the position corresponding to TGA in the gene confirmed that UGA is translated as selenocysteine. The presence of cysteine followed by a reactive selenocysteine residue in this C-terminal region of the protein may explain some of the unusual properties of the mammalian TRs.
Liu
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Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 108, Bethesda, MD 20892, USA.
Stadtman
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TC
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms; D010446:Peptide Fragments / Q000737:chemistry; D010920:Placenta / Q000201:enzymology; D012643:Selenium / Q000032:analysis*; D017279:Selenocysteine / Q000032:analysis*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D017279:Selenocysteine; D005182:Flavin-Adenine Dinucleotide; D006493:Heparin; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium