Human placental steroid-sulfatase solubilized with a cholic-acid derivative: molecular mass, kinetic properties and susceptibility to glycosidases.

L Dibbelt, E Kuss
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引用次数: 14

Abstract

Human placental steroid-sulfatase was extracted nearly quantitatively from microsomes as well as from acetone dry powder of placenta homogenates using CHAPS as detergent. The solubilized enzyme was enriched 10-fold by ammonium sulfate precipitation and gel chromatography. The sulfatase extracted from both microsomes and acetone dry powder eluted as a single fraction on Sepharose 6B, but with different apparent molecular masses (390 and 270 kDa, respectively). Kinetic experiments with the sulfate esters of dehydroepiandrosterone, 16 alpha-hydroxydehydroepiandrosterone, estrone, and estriol as substrates or inhibitors indicated that the solubilized sulfatase was fully active. Both the particulate and the extracted enzyme showed higher affinities for the 16-unsubstituted than for the 16 alpha-hydroxylated substrates. Whereas a competitive inhibition was observed in mixed substrate incubations with dehydroepiandrosterone sulfate/16 alpha-hydroxydehydroepiandrosterone sulfate and estrone sulfate/estriol sulfate, diverse patterns of inhibition were obtained with dehydroepiandrosterone sulfate/estrone sulfate, depending on the sulfatase preparation used. However, evidence for the distinct nature of the steroid-sulfatase and the estrogen-sulfatase was not obtained. The membrane-bound, but not the solubilized enzyme was to a certain degree sensitive to lipase and acetone. The solubilized sulfatase strongly bound to ConA-Sepharose. This observation together with the elution by alpha-methyl mannoside were indicative of the presence of carbohydrates on the sulfatase. Since its enzymatic activity was markedly decreased by the effects of alpha-mannosidase and N-acetylglucosaminidase, a possible involvement of the carbohydrate moiety in the catalytic activity of the sulfatase might be considered.

用胆酸衍生物溶解的人胎盘类固醇硫酸酯酶:分子质量、动力学性质和对糖苷酶的敏感性。
用CHAPS作为洗洁剂,从胎盘微粒体和胎盘匀浆丙酮干粉中几乎定量地提取了人胎盘类固醇硫酸酯酶。经硫酸铵沉淀法和凝胶层析,可使酶富集10倍。从小体和丙酮干粉中提取的磺化酶在Sepharose 6B上作为单一组分洗脱,但表观分子质量不同(分别为390和270 kDa)。以脱氢表雄酮硫酸盐酯、16 α -羟脱氢表雄酮、雌酮和雌三醇为底物或抑制剂的动力学实验表明,可溶性硫酸酯酶具有充分的活性。颗粒和提取的酶对16-未取代物的亲和力高于对16-羟基化底物的亲和力。虽然在硫酸脱氢表雄酮/16 α -羟基硫酸脱氢表雄酮和硫酸雌酮/硫酸雌三醇混合底物培养中观察到竞争性抑制,但在硫酸脱氢表雄酮/硫酸雌酮混合底物培养中,根据所使用的硫酸酯酶制剂的不同,获得了不同的抑制模式。然而,没有证据表明类固醇-硫酸酯酶和雌激素-硫酸酯酶具有不同的性质。膜结合酶对脂肪酶和丙酮有一定的敏感性,而溶解酶对脂肪酶和丙酮不敏感。可溶性硫酸酯酶与ConA-Sepharose紧密结合。这一观察结果与α -甲基甘露糖苷的洗脱表明,在硫酸盐酶上存在碳水化合物。由于α -甘露糖苷酶和n -乙酰氨基葡萄糖苷酶的作用使其酶活性明显降低,因此可以认为碳水化合物部分可能参与了硫酸盐酶的催化活性。
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