Venkatesan S, Gershowitz A, Moss B
J Biol Chem. 1980 Apr 10;255(7):2829-34.
mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.
信使核糖核酸鸟苷酸转移酶，一种催化鸟苷酸残基从鸟苷三磷酸转移至核糖核酸5'端以形成被鉴定为G(5')pppN-的帽状结构的酶，已从人宫颈癌细胞（HeLa细胞）核中分离出来。该酶已被纯化约1000倍，并通过柱色谱法（使用二乙氨基乙基纤维素、磷酸纤维素、汽巴蓝琼脂糖和鸟苷三磷酸琼脂糖）与多种其他活性物质分离，包括核糖核酸三磷酸酶和信使核糖核酸（鸟嘌呤-7）甲基转移酶。反应产物通过其对青霉核酸酶和碱性磷酸酶的抗性、对蛇毒磷酸二酯酶的敏感性以及相对于标准品的电泳和色谱迁移率来鉴定。在pH 7.5、存在锰离子或镁离子、鸟苷三磷酸以及合适的受体多聚核糖核苷酸的情况下可获得最佳酶活性。该酶受到高浓度盐和巯基结合试剂的抑制，但不受S-腺苷甲硫氨酸或S-腺苷同型半胱氨酸的影响。通过对纯化酶进行蔗糖梯度离心法估计其分子量为48,500。
