Olson H M, Nicholson A W, Cooperman B S, Glitz D G
J Biol Chem. 1985 Aug 25;260(18):10326-31.
Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.
先前的研究工作（Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797 - 3808）表明，[3H]对叠氮嘌呤霉素进行光亲和标记了70S大肠杆菌核糖体，并且光掺入50S亚基蛋白质的顺序为L23大于L18/22大于L15。在本研究中，我们报道了对由对叠氮嘌呤霉素修饰的50S亚基与针对该抗生素的N6,N6 - 二甲基腺苷部分的抗体形成的复合物进行的免疫电子显微镜研究。在电子显微镜照片中，对叠氮嘌呤霉素修饰的50S亚基看起来与未修饰的对照亚基相同。在显微镜照片中观察到了修饰亚基与针对对叠氮嘌呤霉素的N6,N6 - 二甲基腺苷部分的抗体形成的复合物。分别评估了带有单个结合抗体的单个亚基（单体复合物）和由单个抗体交联的亚基对（二聚体复合物），并显示出相似的结果。确定了对叠氮嘌呤霉素光掺入的两个区域。主要位点见于约75%的复合物中，位于中央突起和小突起之间，在远离L7/L12臂的一侧，在一个被认为包含肽基转移酶中心的区域。次要位点意义不明，位于亚基基部距臂最远的位置。这些位置与我们在嘌呤霉素光掺入分析中观察到的位置基本相同（Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649 - 2656），并且在数量上与单体嘌呤霉素 - 50S亚基复合物的评估结果相似。这些数据支持蛋白质L23、L18/22和L15位于主要位点的肽基转移酶中心或其附近，此外还表明次要位点包括一个真正的嘌呤霉素亲和区域。
