A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity

Takashi Tamura, Thressa C. Stadtman

Research output: Contribution to journalArticle

435 Citations (Scopus)

Abstract

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 μM [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 K(m) values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 μM, 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.

Original languageEnglish
Pages (from-to)1006-1011
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number3
DOIs
Publication statusPublished - Feb 6 1996

Keywords

  • flavoprotein
  • selenium-75
  • selenocysteine
  • selenoenzyme

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity'. Together they form a unique fingerprint.

  • Cite this