PURIFICATION AND CHARACTERIZATION OF HUMAN-LIVER DEHYDROEPIANDROSTERONE SULFOTRANSFERASE
PURIFICATION AND CHARACTERIZATION OF HUMAN-LIVER DEHYDROEPIANDROSTERONE SULFOTRANSFERASE
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DOI：
10.1042/bj2600641
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发表时间：
1989-06-15
影响因子：
4.1
通讯作者：
KALB, JM
中科院分区：
文献类型：
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作者：
FALANY, CN;VAZQUEZ, ME;KALB, JM
从人肝细胞液中纯化出一种能硫酸化脱氢表雄酮和其它甾体的硫转移酶。用DEAE-Sepharose CL-6 B和腺苷3“，5”-二磷酸-琼脂糖亲和层析纯化了脱氢表雄酮硫酸转移酶，纯化倍数为621倍。在亲和层析过程中，采用腺苷3“-磷酸5”-磷酸硫酸盐梯度，可将脱氢表雄酮硫酸化活性与苯酚硫酸转移酶催化的对硝基苯酚硫酸化活性区分开来。纯化的酶对脱氢表雄酮最有活性，但能够缀合许多其它类固醇，包括双烯醇酮、雄酮和β-环己基甾酮。雌二醇没有活动对对硝基苯酚或多巴胺，底物的苯酚磺基转移酶，观察到与纯酶。SDS/聚丙烯酰胺凝胶电泳后，考马斯亮蓝染色观察到一个单一的带与亚基分子量为35 kDa的纯化酶。通过Sephacryl S-200上的层析计算出该酶的活性形式的分子量为68-70 kDa，表明该酶的活性形式是二聚体。
A form of sulphotransferase capable of sulphating dehydroepiandrosterone and other steroids was purified from cytosol prepared from human liver. Dehydroepiandrosterone sulphotransferase was purified 621-fold when compared with the activity in cytosol using DEAE-Sepharose CL-6B and adenosine 3'',5''-bisphosphate-agarose affinity chromatography. During affinity chromatography, dehydroepiandrosterone suplhation activity could be resolved from p-nitrohenol sulphation activity catalysed by phenol sulphotransferase by using a gradient of adenosine 3''-phosphate 5''-phosphosulphate. The purified enzyme was most active towards dehydroepiandrosterone but was capable of conjugating a number of other steroids, including pregnenolone, androsterone and .beta.-oestradiol. No activity towards p-nitrophenol or dopamine, substrates for the phenol sulphotransferase, was observed with the pure enzyme. A single band with a subunit molecular mass of 35 kDa was observed by Coomassie Blue staining following SDS/polyacrylamide-gel electrophoresis of the purified enzyme. A molecular mass of 68-70 kDa was calculated for the active form of the enzyme by chromatography on Sephacryl S-200, suggesting that the active form of the enzyme is a dimer.