Evans R K, Haley B E, Roth D A
J Biol Chem. 1985 Jun 25;260(12):7800-4.
We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.
我们使用了光亲和类似物8-叠氮腺苷5'-三磷酸（8-N3ATP）和8-叠氮鸟苷5'-三磷酸（8-N3GTP）来研究烟草花叶病毒（TMV）感染的烟草中一种病毒诱导蛋白（分子量 = 120,000）与TMV诱导的RNA依赖性RNA聚合酶活性之间的关系。当放射性类似物[γ-32P]8-N3ATP和[γ-32P]8-N3GTP与来自TMV感染植物的烟草组织匀浆一起孵育时，在紫外光存在下，标记物会掺入到病毒诱导蛋白中。发现这种掺入完全依赖于紫外光照，并且Mg2+能大大增强这种掺入。光掺入标记物的饱和表明8-N3ATP和8-N3GTP的表观解离常数（Kd）分别为16微摩尔（±3微摩尔）和12微摩尔（±3微摩尔）。用各种非放射性核苷酸和核苷对[γ-32P]8-N3ATP和[γ-32P]8-N3GTP的光标记进行保护，结果表明光标记位点受到核苷三磷酸的保护效果最佳。在200微摩尔浓度下，脱氧核糖核苷三磷酸和核糖核苷三磷酸在保护该位点免受光标记方面都非常有效。这些数据表明，光标记的蛋白可能是RNA依赖性RNA聚合酶的一部分。本文还讨论了核苷酸光亲和类似物作为研究病毒诱导的核苷酸结合蛋白的一种方法的实用性。
