Tolan D R, Hershey J W, Traut R T
Biochimie. 1983 Jul;65(7):427-36. doi: 10.1016/s0300-9084(83)80062-5.
Complexes of purified 40S ribosomal subunits and initiation factor 3 from rabbit reticulocytes were crosslinked using the reversible protein crosslinking reagent, 2-iminothiolane, under conditions shown previously to lead to the formation of dimers between 40S proteins but not higher multimers. The activity of both the 40S subunits and initiation factor 3 was maintained. Protein crosslinked to the factor was purified by sucrose density gradient centrifugation following nuclease digestion of the ribosomal subunit: alternatively, the total protein was extracted from 40S: factor complexes. The protein obtained by either method was analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Ribosomal proteins were found in multimeric complexes of high molecular weight due to their crosslinking to components of eIF3. Identification of the ribosomal proteins appearing below the diagonal was accomplished by elution, radioiodination, two-dimensional polyacrylamide/urea gel electrophoresis, and radioautography. Proteins S2, S3, S3a, S4, S5, S6, S8, S9, S11, S12, S14, S15, S16, S19, S24, S25, and S26 were identified. Because many of the proteins in this group form crosslinked dimers with each other, it was impossible to distinguish proteins directly crosslinked to eIF3 from those crosslinked indirectly through one bridging protein. The results nonetheless imply that the 40S ribosomal proteins identified are at or near the binding site for initiation factor 3.
使用可逆蛋白质交联试剂2-亚氨基硫杂环戊烷，在先前已证明可导致40S蛋白质之间形成二聚体而非更高聚体的条件下，对来自兔网织红细胞的纯化40S核糖体亚基和起始因子3复合物进行交联。40S亚基和起始因子3的活性均得以维持。在对核糖体亚基进行核酸酶消化后，通过蔗糖密度梯度离心法纯化与该因子交联的蛋白质：或者，从40S：因子复合物中提取总蛋白质。通过二维对角线聚丙烯酰胺/十二烷基硫酸钠凝胶电泳分析通过任一方法获得的蛋白质。由于核糖体蛋白与eIF3的组分交联，因此在高分子量的多聚体复合物中发现了它们。通过洗脱、放射性碘化、二维聚丙烯酰胺/尿素凝胶电泳和放射自显影来鉴定出现在对角线下方的核糖体蛋白。鉴定出了蛋白质S2、S3、S3a、S4、S5、S6、S8、S9、S11、S12、S14、S15、S16、S19、S24、S25和S26。由于该组中的许多蛋白质彼此形成交联二聚体，因此无法直接区分与eIF3直接交联的蛋白质和通过一种桥接蛋白间接交联的蛋白质。尽管如此，结果表明所鉴定的40S核糖体蛋白位于起始因子3的结合位点处或附近。
