Falany C N, Krasnykh V, Falany J L
Department of Pharmacology and Toxicology, University of Alabama at Birmingham 35294, USA.
J Steroid Biochem Mol Biol. 1995 Jun;52(6):529-39. doi: 10.1016/0960-0760(95)00015-r.
A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.
利用聚合酶链反应（PCR）技术，从人肝λZap cDNA文库中分离出一种独特的人雌激素硫酸转移酶（hEST-1）cDNA。该酶活性蛋白已在两种细菌表达系统中表达，并对其动力学和免疫学特性进行了表征。hEST-1的全长cDNA长度为994个碱基对，编码一个294个氨基酸的蛋白质，计算分子量为35,123道尔顿。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，纯化的hEST-1迁移时表观分子量为35,000道尔顿。用兔抗hEST-1抗体对大肠杆菌中表达的hEST-1进行免疫印迹分析，得到一条约35,000道尔顿的条带。抗hEST-1抗体在人肝和空肠胞浆中也检测到一条单一的条带，其迁移分子量与表达的hEST-1相同。免疫印迹分析显示，hEST-1与兔抗人酚类硫酸转移酶（hP-PST）或兔抗人脱氢表雄酮硫酸转移酶（hDHEA-ST）抗体也无交叉反应。hEST-1在细菌中表达并纯化至同质。与其他结合雌激素的人硫酸转移酶相比，表达的hEST-1活性对雌激素硫酸化具有显著更高的亲和力。hEST-1在20 nM浓度下对β-雌二醇和雌酮进行最大程度的硫酸化。hEST-1也能对脱氢表雄酮、孕烯醇酮、乙炔雌二醇和1-萘酚进行硫酸化，但所需浓度显著更高；然而，皮质醇、睾酮和多巴胺则不会被硫酸化。本文给出的结果描述了一种不同于其他硫酸化雌激素的人硫酸转移酶的表达和特性。hEST-1对雌激素的高亲和力表明，这种硫酸转移酶在雌激素代谢及其活性调节中可能都很重要。
