Human kidney thiopurine methyltransferase. Photoaffinity labeling with S-adenosyl-L-methionine.
Human kidney thiopurine methyltransferase. Photoaffinity labeling with S-adenosyl-L-methionine.
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DOI：
10.1016/0006-2952(92)90416-g
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发表时间：
1992-08
影响因子：
5.8
通讯作者：
J. van Loon;C. Szumlanski;R. Weinshilboum
中科院分区：
文献类型：
--
作者：
J. van Loon;C. Szumlanski;R. Weinshilboum
硫嘌呤甲基转移酶 (TPMT) 催化杂环和芳香族硫氢基化合物的 S-甲基化，例如硫嘌呤药物 6-巯基嘌呤 (6-MP)。人体组织中的TPMT活性受共同的遗传多态性调节，TPMT活性的“药代”变异是硫嘌呤药物代谢、毒性和疗效个体差异的重要因素。人肾组织含有 TPMT 的两种同工酶，Peak I 和 Peak II，可以通过离子交换色谱分离。我们的实验旨在确定 TPMT 反应的甲基供体 S-腺苷-L-甲硫氨酸 (Ado-Met) 是否可以用作这些同工酶的光亲和配体，作为研究 TPMT 遗传多态性分子基础的第一步。当 [3 H-甲基] Ado-Met 和人肾 TPMT 任一同工酶的部分纯化制剂暴露于 254 nm 的紫外线，然后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳时，35 kDa 的蛋白质是被放射性标记的主要种类。相同的 35 kDa 蛋白质用 [14 C-羧基] Ado-Met 进行光亲和标记，表明标记涉及 Ado-Met 的共价结合而不是蛋白质的甲基化。在连续的 DEAE 离子交换、凝胶过滤和羟基磷灰石层析过程中，TPMT 酶活性与 35 kDa 蛋白质共洗脱。 TPMT 酶活性抑制剂（包括 S-腺苷-L-同型半胱氨酸、sinefungin、6-甲硫基嘌呤和 3, 4-二甲氧基-5-羟基苯甲酸）以浓度依赖性方式抑制 TPMT Peak I 和 Peak II 同工酶制剂中 35 kDa 蛋白的光亲和标记。 6-MP，TPMT 反应的甲基受体底物。所有这些结果都与 35 kDa 蛋白是 TPMT 的结论相一致。用 [3 H] Ado-Met 对 TPMT 进行光亲和标记，可以将酶纯化至均质，并研究其活性位点处或附近的氨基酸序列。
Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of heterocyclic and aromatic sulfhydryl compounds such as the thiopurine drug 6-mercaptopurine (6-MP). TPMT activity in human tissue is regulated by a common genetic polymorphism, and “pharmacogenetic” variation in TPMT activity is an important factor in individual differences in thiopurine drug metabolism, toxicity and therapeutic efficacy. Human renal tissue contains two isozymes of TPMT, Peak I and Peak II, that can be separated by ion exchange chromatography. Our experiments were performed to determine whether S-adenosyl-L-methionine (Ado-Met), the methyl donor for the TPMT reaction, could be used as a photoaffinity ligand for these isozymes as one step in the study of the molecular basis for the TPMT genetic polymorphism. When [3 H-methyl] Ado-Met and partially purified preparations of either isozyme of human kidney TPMT were exposed to ultraviolet light at 254 nm, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35 kDa protein was the predominant species that was radioactively labeled. The same 35 kDa protein was photoaffinity labeled with [14 C-carboxyl] Ado-Met, demonstrating that labeling involved covalent binding of Ado-Met rather than methylation of the protein. TPMT enzymatic activity co-eluted with the 35 kDa protein during sequential DEAE ion exchange, gel filtration and hydroxylapatite chromatography. Inhibitors of TPMT enzymatic activity including S-adenosyl-L-homocysteie, sinefungin, 6-methylmercaptopurine and 3, 4-dimethoxy-5-hydroxybenzoic acid inhibited photoaffinity labeling of the 35 kDa protein in preparations of both TPMT Peak I and Peak II isozymes in a concentration-dependent fashion, as did 6-MP, the methyl acceptor substrate for the TPMT reaction. All of these results were compatible with the conclusion that the 35 kDa protein was TPMT. Photoaffinity labeling of TPMT with [3 H] Ado-Met should make it possible to purify the enzyme to homogeneity and to study amino acid sequences at or near its active site.