Sakakibara Y, Takami Y, Zwieb C, Nakayama T, Suiko M, Nakajima H, Liu M C
Department of Biochemistry, University of Texas Health Center at Tyler 75710, USA.
J Biol Chem. 1995 Dec 22;270(51):30470-8. doi: 10.1074/jbc.270.51.30470.
A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000. Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.
通过五步柱色谱法（羟基磷灰石I、DEAE生物凝胶、ATP琼脂糖I、羟基磷灰石II和ATP琼脂糖II）从大鼠肝脏胞质溶胶中纯化出一种新型磺基转移酶，达到电泳纯。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化酶的最小分子量约为33,000。凝胶过滤色谱显示其天然分子量约为34,000，表明该酶以单体形式存在。纯化的磺基转移酶对各种酪氨酸和3,4-二羟基苯丙氨酸（多巴）异构体（除DL-邻酪氨酸外）表现出酶活性，最适pH为9.25。甲状腺激素以及多巴胺和对硝基苯酚也可作为底物。在3'-磷酸腺苷5'-磷酸硫酸酯浓度恒定为14μM时，该酶（命名为多巴/酪氨酸磺基转移酶）对L-多巴的表观Km值为0.76 mM。对完整酶进行N端测序时发现其N端被封闭。通过分析其蛋白水解片段获得的三个内部部分氨基酸序列与其他已知大鼠肝脏磺基转移酶的同源序列不同。从大鼠肝脏cDNA文库中分离出的全长cDNA推导的氨基酸序列证实，多巴/酪氨酸磺基转移酶是一种新型芳基磺基转移酶。用携带全长cDNA的表达载体（pMSG-CMV）转染COS-7细胞后，表达出一种33 kDa的蛋白，其酶学和免疫学性质与纯化的多巴/酪氨酸磺基转移酶相似。
