Mizumoto K, Lipmann F
Proc Natl Acad Sci U S A. 1979 Oct;76(10):4961-5. doi: 10.1073/pnas.76.10.4961.
Rat liver nuclei were isolated and sonicated for extraction in order to study the capping of RNA. The guanosine 7-methyltransferase was purified from the extract by hydroxylapatite column chromatography with stepwise addition of phosphate buffer. It was assayed by using as methyl acceptor synthetic G(5')ppp(5')G and S-adenosylmethionine as donor. The enzyme appeared in a sharp peak at 160 mM. The same peak fraction was subsequently found to contain the enzyme that guanylylates short synthetic polynucleotides and low molecular weight yeast RNA as acceptors. The two enzymatic activities were separated on Sephadex G-150 chromatography, yielding guanylyltransferase and guanosine 7-methyltransferase with molecular weights of approximately 65,000 and 130,000 respectively. Guanylyltransferase was further purified by CM-Sephadex chromatography, whereby G-7-methyltransferase was completely removed. Dithiothreitol was essential for guanylylation, and 2 mM Mn2+ (optimum) was twice as active as 8 mM Mg2+ (optimum). The alpha-32P of [32P]GTP, but not its beta- or gamma-32P was incorporated into the cap structure. By using unlabeled GTP with [beta-32P]ppGpCpC-poly(A2,U2,G) as acceptor, [beta'-32P]-GpppG... was formed. Our purified transguanylylation enzyme was found to catalyze a [32P]pyrophosphate exchange with GTP, which may be useful as a rapid assay for transguanylylation.
为了研究RNA的加帽过程，分离并超声处理大鼠肝脏细胞核以进行提取。通过羟基磷灰石柱色谱法，逐步添加磷酸盐缓冲液，从提取物中纯化鸟苷7-甲基转移酶。使用合成的G(5')ppp(5')G作为甲基受体，S-腺苷甲硫氨酸作为供体来测定该酶。该酶在160 mM处出现一个尖锐的峰。随后发现同一峰级分中含有以短合成多核苷酸和低分子量酵母RNA作为受体进行鸟苷酸化的酶。通过Sephadex G-150色谱法分离这两种酶活性，得到分子量分别约为65,000和130,000的鸟苷酸转移酶和鸟苷7-甲基转移酶。通过CM-Sephadex色谱法进一步纯化鸟苷酸转移酶，从而完全去除G-7-甲基转移酶。二硫苏糖醇对于鸟苷酸化至关重要，2 mM Mn2+（最佳浓度）的活性是8 mM Mg2+（最佳浓度）的两倍。[32P]GTP的α-32P而非其β-或γ-32P被掺入帽结构中。使用未标记的GTP与[β-32P]ppGpCpC-poly(A2,U2,G)作为受体，形成了[β'-32P]-GpppG...。我们纯化的转鸟苷酸化酶被发现可催化与GTP的[32P]焦磷酸交换，这可能作为转鸟苷酸化的一种快速测定方法。
