Cho J M, Kimball A P
Biochem Pharmacol. 1982 Aug 15;31(16):2575-81. doi: 10.1016/0006-2952(82)90703-1.
9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.
9-β-D-阿拉伯呋喃糖基-6-巯基嘌呤（ara-6-MP）用于亲和标记小麦胚芽DNA依赖性RNA聚合酶II（或B）（核苷三磷酸：RNA核苷酸转移酶，EC 2.7.7.6）。发现这种核苷类似物在[3H]UMP掺入方面是一种竞争性抑制剂。当酶与过量浓度的底物预孵育时，天然底物可保护酶不被ara-6-MP灭活，这表明抑制剂结合在延伸亚位点。抑制剂以0.6:1的化学计量比结合酶的催化中心。巯基试剂二硫苏糖醇可逆转ara-6-MP的抑制作用，这表明抑制剂的6-硫醇基团与酶催化中心的一个必需半胱氨酸残基紧密相互作用。RNA聚合酶II-ara-6-MP复合物的链霉蛋白酶消化产物的色谱分析也表明ara-6-MP结合了一个半胱氨酸残基。变性的[6-35S]ara-6-MP标记的RNA聚合酶II的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，超过80%的放射性与该酶的IIb亚基相关。
