Purification and characterization of rat liver minoxidil sulphotransferase.
Purification and characterization of rat liver minoxidil sulphotransferase.
复制标题
大鼠肝脏米诺地尔磺基转移酶的纯化和表征。
DOI：
10.1042/bj2700721
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Falany,CN
中科院分区：
文献类型：
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作者：
Hirshey,SJ;Falany,CN
米诺地尔（Mx）是一种嘧啶N-氧化物，在治疗上用作抗高血压药，并用于诱导男性型脱发患者的毛发生长。Mx NO-硫酸盐被认为是产生这些效应的活性剂。本文介绍了一种独特的硫转移酶（ST）的纯化，从大鼠肝胞质溶胶，能够催化Mx的硫酸化。经DEAE-SepharoseCL-6B柱层析、羟基磷灰石柱层析和ATP-琼脂糖亲和柱层析，Mx-ST活性比胞液中的活性纯化了240倍。纯化的酶也能够在低浓度（小于10 μ M）下硫酸化对硝基苯酚（PNP）。通过SDS/PAGE和反相h.p.l.c.评价，将Mx-ST纯化至均一。用凝胶排阻色谱法测得该酶的活性分子量为66，000~68，000 Da，亚基分子量为35，000 Da，对Mx、3 ′-磷酸腺苷、5 ′-磷酸硫酸和PNP的表观Km值分别为625 μ M、5.0 μ M和0.5 μ M。然而，PNP在高于1.2 μ M的浓度下显示出有效的底物抑制。在兔中产生的纯酶抗体在大鼠肝胞质溶胶中检测到一条带，亚基分子量为35，000 Da，通过免疫印迹法测定。抗（大鼠Mx-ST）抗体也与人肝酚磺基转移酶的苯酚硫酸化形式反应，表明这些蛋白质之间存在一些结构相似性。
Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3′-phosphoadenosine 5′-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.