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首页> 外文学位 >Purification, molecular cloning, and expression of rat liver minoxidil sulfotransferase.
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Purification, molecular cloning, and expression of rat liver minoxidil sulfotransferase.

机译：大鼠肝脏米诺地尔磺基转移酶的纯化，分子克隆和表达。

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摘要

A unique phenol sulfotransferase (PST) isozyme, minoxidil ST (Mx-ST), catalyzes the conversion of Mx to Mx-sulfate (Mx-S). Mx-S has been determined to be the active agent in the vasodilatory and hair growth stimulatory effects of the drug. Mx-ST has been purified approximately 240-fold from male rat liver cytosol to apparent homogeneity by anion-exchange, hydroxylapatite, and ATP-agarose affinity chromatography. Purified Mx-ST is capable of sulfating Mx, a pyrimidine N-oxide, as well as the phenolic substrate, 4-nitrophenol (PNP). The apparent Kms of Mx-ST for Mx, PAPS, and PNP were 625 {dollar}mu{dollar}M, 5.0 {dollar}mu{dollar}M, and 0.5 {dollar}mu{dollar}M, respectively. Mx-ST displayed substrate inhibition at concentrations above 1.2 {dollar}mu{dollar}M PNP and 1 mM Mx.; Mx-ST exists as a homodimer of two 35 kDa subunits in its active form. Polyclonal antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35 kDa as determined by immunoblotting. Furthermore, this antibody has been used in immunolocalization studies of rat anagen pelage and vibrissae follicles. Localization of the enzyme in the hair follicle suggests that the outer root sheath may be an active site of Mx sulfation and in some way mediates its hair growth effects.; A cDNA encoding Mx-ST has been isolated from a {dollar}lambda{dollar}gtll cDNA library constructed from poly (A)+ RNA prepared from female Sprague-Dawley rat liver. The largest cDNA, designated Mx-STb, consists of 1245 base pairs and contains an open reading frame of 291 amino acids. The size of the protein obtained by in vitro translation of Mx-STb is identical to that of the pure protein. Results of initial studies of the expression of Mx-STb in COS-1 cells indicate that the expressed protein displays characteristic Mx-ST and PNP-ST activity, is recognized by rabbit polyclonal antibodies raised against pure rate liver Mx-ST, and migrates at approximately 35,000 daltons during SDS-PAGE.; Mx-ST is most likely the homologous enzyme to human liver phenol ST, P-PST. Antibodies raised to rat liver Mx-ST and human PST cross-react with purified P-PST and Mx-ST, respectively, suggesting the enzymes are structurally related. In addition, Mx-ST and P-PST exhibit similar substrate specificities. Finally, a high percentage of nucleotide and amino acid sequence identity (80%) is observed between these two enzymes. (Abstract shortened with permission of author.)
机译：独特的苯酚磺基转移酶（PST）同功酶，米诺地尔ST（Mx-ST）催化Mx转化为Mx-硫酸盐（Mx-S）。已经确定Mx-S是药物在血管舒张和毛发生长刺激作用中的活性剂。已通过阴离子交换，羟基磷灰石和ATP-琼脂糖亲和色谱法从雄性大鼠肝细胞溶质中纯化了Mx-ST约240倍，使其具有明显的均质性。纯化的Mx-ST能够硫酸化Mx（嘧啶N-氧化物）以及酚类底物4-硝基苯酚（PNP）。 Mx，PAPS和PNP的Mx-ST的表观Kms分别为625μM，5.0μM和0.5μM。 Mx-ST在高于1.2μM{M} PNP和1mM Mx的浓度下显示底物抑制。 Mx-ST以其活性形式以两个35 kDa亚基的同型二聚体形式存在。在兔体内产生的针对纯酶的多克隆抗体通过免疫印迹法检测到大鼠肝细胞质溶胶中的一条条带，其亚基分子量为35 kDa。此外，该抗体已用于大鼠毛细血管原性和触须卵泡的免疫定位研究。酶在毛囊中的定位表明，外根鞘可能是Mx硫酸化的活性部位，并以某种方式介导了其毛发生长作用。已经从由雌性Sprague-Dawley大鼠肝脏制备的poly（A）+ RNA构建的{λ}λ{gt} gtll cDNA文库中分离了编码Mx-ST的cDNA。最大的cDNA，称为Mx-STb，由1245个碱基对组成，并包含291个氨基酸的开放阅读框。通过体外翻译Mx-STb获得的蛋白质大小与纯蛋白质大小相同。 COS-1细胞中Mx-STb表达的初步研究结果表明，表达的蛋白显示出特征性的Mx-ST和PNP-ST活性，可被抗纯率肝Mx-ST的兔多克隆抗体识别，并在在SDS-PAGE期间约为35,000道尔顿。 Mx-ST最有可能与人肝酚ST，P-PST同源。针对大鼠肝脏Mx-ST和人PST的抗体分别与纯化的P-PST和Mx-ST交叉反应，表明这些酶在结构上相关。此外，Mx-ST和P-PST表现出相似的底物特异性。最后，在这两种酶之间观察到高百分比的核苷酸和氨基酸序列同一性（80％）。 （摘要经作者许可缩短。）

著录项

  • 作者

    Hirshey, Sharon Joan.;

  • 作者单位

    The University of Rochester.;

  • 授予单位 The University of Rochester.;
  • 学科 Health Sciences Pharmacology.
  • 学位 Ph.D.
  • 年度 1992
  • 页码 195 p.
  • 总页数 195
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 药理学;
  • 关键词

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