A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity.
We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.
Tamura
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Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0320, USA.
Stadtman
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D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007700:Kinetics; D008175:Lung Neoplasms; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D012643:Selenium / Q000378:metabolism; D051149:Selenoprotein P; D051140:Selenoproteins; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium