PURIFICATION AND CHARACTERIZATION OF HUMAN LIVER PHENOL-SULFATING PHENOL SULFOTRANSFERASE
PURIFICATION AND CHARACTERIZATION OF HUMAN LIVER PHENOL-SULFATING PHENOL SULFOTRANSFERASE
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DOI：
10.1016/0003-9861(90)90265-z
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发表时间：
1990-05-01
影响因子：
3.9
通讯作者：
ROTH, JA
中科院分区：
文献类型：
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作者：
FALANY, CN;VAZQUEZ, ME;ROTH, JA
用DEAE-纤维素、Sephacryl S-200和3“-5”-二磷酸腺苷-琼脂糖亲和层析从人肝胞液中纯化并鉴定了苯酚硫酸化形式的苯酚磺基转移酶（P-PST）。在纯化过程中，P-PST从苯酚磺基转移酶（M=PST）和脱氢表雄酮磺基转移酶的单胺硫酸化形式中分离出来，这两种酶也存在于人肝细胞溶质中。P-PST活性纯化560倍，肝细胞质相比，纯化的酶具有340 nmol硫酸苯酚每分钟每毫克蛋白质的比活性。经Sephacryl S-200柱层析测定，P-PST的表观分子量为68，000 Da，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，P-PST的亚基分子量为32，000 Da，表明P-PST在体内以同源二聚体形式存在。抗人血小板M-PST的抗体与纯P-PST有强烈的交叉反应，表明这两种PST在结构上密切相关。两种类型的P-PST活动已被确定在不同的人的肝脏，其热稳定性和洗脱过程中的阴离子交换色谱。每一个肝脏检查拥有只有一种类型的P-PST活动。两种P-PST均具有相同的亚基分子量和免疫反应活性，但两种P-PST的热稳定性差异与肝胞液的制备方法有关。两种类型的P-PST活性通过与50 μ M N-乙基马来酰亚胺或5 mM苯乙二醛温育被抑制到相似的程度。这些结果表明，不同人类肝脏中的两种类型的P-PST非常相似，并且可能代表该酶的不同等位基因形式。
The phenol-sulfating form of phenol sulfotransferase (P-PST) was purified and characterized from human liver cytosol using DEAE-cellulose, Sephacryl S-200, and 3''-5''-diphosphoadenosine-agarose affinity chromatography. During the purification procedure, P-PST was resolved from the monoamine-sulfating form of phenol sulfotransferase (M=PST) and dehydroepiandrosterone sulfotransferase, which are also present in human liver cytosol. P-PST activity was purified 560-fold as compared to liver cytosol and the purified enzyme possessed a specific activity of 340 nmol phenol sulfated per minute per milligram protein. Enzymatically active P-PST has an apparent molecular size of 68,000 Da as determined by Sephacryl S-200 chromatography and a subunit molecular weight of 32,000 Da as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, suggesting that P-PST exists in vivo as a homodimer. Antibodies raised to human platelet M-PST cross-reacted strongly with pure P-PST suggesting the two PSTs are structurally closely related. Two types of P-PST activity have been identified in different human livers by their thermostability and elution during anion-exchange chromatography. Each of the livers examined posssessed only one type of P-PST activity. Both types of P-PST were shown to possess the same subunit molecular weight and immunonoreactivity, whereas the differences in thermosabiltiy of the two P-PST activities appeared to be related to the method of preparation of liver cytosol. Both types of P-PST activity were inhibited to similar extents by incubation with 50 .mu.M N-ethylmaleidmide or 5 mM phenylglyoxal. These results suggest that the two types of P-PST in different human livers are very similar and probably represent different allelic forms of the enzyme.