克隆和表达的人类肝脏UDP-glucuronosyltransferase COS-1细胞。3,4-catechol雌激素和雌三醇为主要基质。
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Ritter JK,辛YY,欧文斯
克隆和表达的人类肝脏UDP-glucuronosyltransferase COS-1细胞。3,4-catechol雌激素和雌三醇为主要基质。
J生物化学杂志。1990年5月15日,265 (14):7900 - 6。
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2159463 (在PubMed]
- 文摘
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人类cDNA克隆,UDPGTh-2、编码肝脏UDP-glucuronosyltransferase(转移酶)隔绝λgt11 cDNA库通过杂交鼠标转移酶cDNA克隆,UDPGTm-1(木村,T。欧文斯,i s(1987)欧元。j . Biochem.168 515 - 521)。这两个克隆编码区核苷酸序列的身份的74%。UDPGTh-2编码529个氨基酸的蛋白质的氨基末端membrane-insertion信号肽和羧基末端membrane-spanning地区。有三个潜在asparagine-linked糖基化网站残留67年,68年和315年。为了建立底物特异性,克隆插入pSVL向量(pUDPGTh-2)和COS-1细胞中表达。转移酶的存在与全等先生52000年在转染细胞培养的[35 s]蛋氨酸与山羊antimouse immunocomplexed所示产品转移酶免疫球蛋白(Mackenzie, p。、Hjelmeland l . H。欧文斯,i s(1984)拱门。物化学。Biophys。231年,487 - 497)和蛋白质A-Sepharose钠十二烷基sulfate-polyacrylamide凝胶电泳分析和放射自显影法。 The transferase is a glycoprotein as indicated by a shift in Mr congruent to 3000-4000 when expressed in the presence of tunicamycin. Sixty potential substrates were tested using cells transfected with pUDPGTh-2. The order of relative substrate activity was as follows: 4-hydroxyestrone greater than estriol greater than 2-hydroxyestriol greater than 4-hydroxyestradiol greater than 6 alpha-hydroxyestriol greater than 5 alpha-androstane-3 alpha,11 beta,17 beta-triol = 5 beta-androstane-3 alpha,11 beta,17 beta-triol. There were only trace amounts of glucuronidation of 2-hydroxyestrone and 2-hydroxyestradiol, and, in contrast to other cloned transferases, no glucuronidation of either the primary estrogens/androgens (estrone, 17 beta-estradiol/testosterone, androsterone) or any of the exogenous substrates tested. A Lineweaver-Burk plot of the effect of 4-hydroxyestrone concentration on the velocity of glucuronidation shows an apparent Km of 13 microM. The unique specificity of this transferase for 3,4-catechol estrogens and estriol suggests it may play an important role in regulating the level and activity of these potent and active estrogen metabolites.