Expression of human liver 3, 4-catechol estrogens UDP-Glucuronosyltransferase cDNA in COS 1 cells.
The human cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT), was isolated from a lambdagt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. The two clones had 74% nucleotide sequence identities in the coding region UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. Sixty potential substrates were tested using cells transfected with pUDPGTh2. The order of relative substrate activity, was as follows: 4-hydroxyestrone > estriol >2-hydroxyestriol > 4-hydroxyestradiol > 6alpha-hydroxyestradiol > 5alpha-androstane-3alpha, 11beta, 17beta-triol=5beta-androstane-3alpha, 11beta, 17beta-triol. There were only trace amounts of glucuronidation of 2-hydroxyestradiol and 2-hydroxyestrone, and in contrast to other cloned transferase, no gulcuronidation of either the primary estrogens and androgens (estrone, 17beta-estradiol/testosterone, androsterone) or any of the exogenous substrates tested was detected. A lineweaver-Burk plot of the effect of 4-hydroxyestrone concentration on the velocity of glucuronidation showed an apparent Km of 13 muM. The unique specificity of this transferase might play an important role in regulating the level and activity of these potent and active estrogen metabolites.
Ahn
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MR
College of Pharmacy, Ewha Womans University, #11-1, Daehyundong, Sudaemunku, 120-750, Seoul, Korea.
Owens
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IS
Sheen
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