Barrieux A, Rosenfeld M G
J Biol Chem. 1977 Jun 10;252(11):3843-7.
Purified GTP-dependent Met-tRNAf binding protein (EIF2) prepared from the 0.5 M KCl eluate of reticulocyte polyribosomes was successfully resolved into its three-component subunits by isoelectric focusing in the presence of urea. The 37,000-dalton subunit focused at a pH of 5.8 and was resolved into two spots; the 48,000-dalton subunit focused at a pH of 6.6 and was resolved into three spots; and the 52,000-dalton subunit exhibited an isoelectric point of 8.9 and migrated as a single spot. When isolated 37,000- and 48,000-dalton subunit was found to possess Met-tRNAf and mRNA binding activities while the 37,000-dalton subunit was found to possess GDP binding activity. Phosphorylation of EIF2 by protein kinases present in reticulocyte lysates was demonstrated using [gamma-32P]GTP or [gamma-32P]ATP as the phosphate donor. The 37,000-dalton subunit was preferentially phosphorylated when [gamma-32P]ATP was used as substrate; the 48,000-dalton subunit was preferentially phosphorylated when the [gamma-52P]GTP was used as phosphate donor, although some phosphorylation of the 37,000-dalton subunit was also observed. The 37,000-dalton subunit of ribosome-associated EIF2 was present predominantly in a dephosphorylated form following purification.
从网织红细胞多核糖体的0.5M KCl洗脱液中制备的纯化的GTP依赖性甲硫氨酸 - tRNAf结合蛋白（EIF2），通过在尿素存在下的等电聚焦成功地分解为其三个组成亚基。37,000道尔顿的亚基在pH 5.8处聚焦并分解为两个斑点；48,000道尔顿的亚基在pH 6.6处聚焦并分解为三个斑点；52,000道尔顿的亚基等电点为8.9，以单个斑点迁移。当分离时，发现37,000和48,000道尔顿的亚基具有甲硫氨酸 - tRNAf和mRNA结合活性，而37,000道尔顿的亚基具有GDP结合活性。使用[γ-32P]GTP或[γ-32P]ATP作为磷酸盐供体，证明了网织红细胞裂解物中存在的蛋白激酶对EIF2的磷酸化作用。当使用[γ-32P]ATP作为底物时，37,000道尔顿的亚基优先被磷酸化；当使用[γ-52P]GTP作为磷酸盐供体时，48,000道尔顿的亚基优先被磷酸化，尽管也观察到37,000道尔顿亚基的一些磷酸化。纯化后，核糖体相关EIF2的37,000道尔顿亚基主要以去磷酸化形式存在。
