Green J M, Singer S S
Can J Biochem Cell Biol. 1983 Jan;61(1):15-22. doi: 10.1139/o83-003.
A radioisotopic assay for cytoplasmic estradiol-17 beta sulfotransferase activity in rat liver was developed. Routine enzyme assays used 120 microM [3H]estradiol-17 beta, 240 microM 3'-phosphoadenosine-5'-phosphosulfate, and enzyme samples containing up to 0.60 mg of cytosol protein. Livers from males and females sulfated 934 +/- 231 and 861 +/- 266 nmol estradiol-17 beta . h-1 . g-1. DEAE-Sephadex A-50 chromatography showed that most of the cytoplasmic enzyme activity eluted as one peak that was well separated from glucocorticoid and 3 beta-hydroxysteroid sulfotransferases. Pooled column fractions containing this estradiol-17 beta sulfotransferase exhibited kinetic properties similar to the enzyme activity in cytosol, but gave slightly greater activity with 180 microM estradiol-17 beta and 360 microM 3'-phosphoadenosine-5'-phosphosulfate. Apparent Km's for the steroid and the coenzyme were 71-85 and 80-93 microM, respectively. The pH optimum for the enzyme reaction was 7.75 +/- 0.25. The enzyme sulfated estradiol-17 beta at all concentrations tested between 10 and 180 microM. It did not sulfate estrone, testosterone, dehydroepiandrosterone, or cortisol well at any test concentration between 10 and 120 microM. The sulfation product was estra-1,3,5-triene-17 beta-ol-3-sulfate. The molecular weight of the enzyme was 54 500 +/- 2300 by Sephadex G-100 chromatography. The estradiol-17 beta sulfotransferase was inhibited strongly by phenols, but not by corticosterone, deoxycorticosterone, dehydroepiandrosterone, estrone, progesterone, or testosterone. Adrenalectomy diminished the estradiol-17 beta sulfotransferase activity greatly, owing to decreases of the specific estradiol-17 beta sulfotransferase concentration. The possible relationships between the specific estradiol-17 beta sulfotransferase and other sulfotransferases in rat liver are discussed.
已开发出一种用于测定大鼠肝脏细胞质中雌二醇 - 17β硫酸转移酶活性的放射性同位素测定法。常规酶测定使用120微摩尔[3H]雌二醇 - 17β、240微摩尔3'-磷酸腺苷 - 5'-磷酸硫酸酯，以及含有高达0.60毫克胞质溶胶蛋白的酶样品。雄性和雌性大鼠肝脏对雌二醇 - 17β的硫酸化速率分别为934±231和861±266纳摩尔·小时-1·克-1。DEAE - 葡聚糖A - 50柱色谱显示，大部分细胞质酶活性以一个峰的形式洗脱，该峰与糖皮质激素和3β - 羟基类固醇硫酸转移酶完全分离。含有这种雌二醇 - 17β硫酸转移酶的合并柱级分表现出与胞质溶胶中酶活性相似的动力学特性，但在180微摩尔雌二醇 - 17β和360微摩尔3'-磷酸腺苷 - 5'-磷酸硫酸酯存在时活性略高。该类固醇和辅酶的表观Km值分别为71 - 85和80 - 93微摩尔。酶反应的最适pH为7.75±0.25。在10至180微摩尔之间的所有测试浓度下，该酶都能使雌二醇 - 17β硫酸化。在10至120微摩尔之间的任何测试浓度下，它对雌酮、睾酮、脱氢表雄酮或皮质醇的硫酸化效果都不佳。硫酸化产物为estra - 1,3,5 - 三烯 - 17β - 醇 - 3 - 硫酸酯。通过葡聚糖G - 100柱色谱法测得该酶的分子量为54500±2300。雌二醇 - 17β硫酸转移酶受到酚类物质的强烈抑制，但不受皮质酮、脱氧皮质酮、脱氢表雄酮、雌酮、孕酮或睾酮的抑制。肾上腺切除术由于特异性雌二醇 - 17β硫酸转移酶浓度降低而使雌二醇 - 17β硫酸转移酶活性大大降低。本文讨论了大鼠肝脏中特异性雌二醇 - 17β硫酸转移酶与其他硫酸转移酶之间可能的关系。
