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.