Benne R, Wong C, Luedi M, Hershey J W
J Biol Chem. 1976 Dec 10;251(23):7675-81.
Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.
起始因子IF-E2是从兔网织红细胞中分离出来的，通过硫酸铵分级沉淀、DEAE-纤维素和磷酸纤维素柱层析，以及在合适的情况下通过蔗糖密度梯度离心，将其纯化了120倍，达到近乎均一的程度。该因子是一种复合蛋白，包含分子量分别为57,000、52,000和36,000的三种不同多肽。在整个纯化过程以及在非变性缓冲液中的聚丙烯酰胺凝胶电泳过程中，它都表现为一种复合物，但在变性缓冲液中电泳时，其三个组分很容易被分离。其任何一个组分都不对应于其他起始因子的任何多肽，也不对应于在高盐浓度缓冲液中洗涤过的核糖体的任何蛋白质。确定这三种多肽的化学计量比为1:1:1；基于每个复合物各有一个拷贝的假设，计算出的因子分子量为145,000，这一数值与测量值160,000一致。起始因子IF-E2在体外通过还原烷基化或用同样从兔网织红细胞中分离出来的蛋白激酶进行磷酸化进行放射性标记。这两种方法都不会导致该因子与GTP和起始甲硫氨酰-tRNA形成三元复合物的能力发生可测量的变化。5'-鸟苷基亚甲基二膦酸可以替代GTP，但仅在相对较高的浓度下。通过蔗糖密度梯度离心研究了标记的起始因子IF-E2和甲硫氨酰-tRNA与40S核糖体亚基的结合。只有当三元复合物的所有三个组分都包含在反应混合物中时，才能看到该因子有明显的结合。添加珠蛋白信使RNA和起始因子IF-E3不会刺激因子或甲硫氨酰-tRNA的结合。结果表明，起始因子IF-E2的所有三个多肽组分都与这些新生的起始复合物结合。
