Feldhoff R C, Karinch A M, Kimball S R, Jefferson L S
Department of Biochemistry, School of Medicine, University of Louisville, KY 40292.
Prep Biochem. 1993 Aug;23(3):363-74. doi: 10.1080/10826069308544562.
Eukaryotic initiation factors 2 and 2B (eIF-2; eIF-2B) are components of the rate-limiting step in the initiation of eukaryotic protein synthesis and are involved in the regulation of this process. When the alpha-subunit of eIF-2 is phosphorylated by an eIF-2 alpha kinase, the phosphorylated eIF-2 alpha (eIF-2 alpha(P)) binds tightly to eIF-2B and prevents the recycling of eIF-2.GDP to eIF-2.GTP which is required for sustained initiation of protein synthesis. The minute quantities of these proteins which are present in rat liver and muscle cytosol along with hundreds of other proteins has hindered purification efforts, as well as structure:function and regulatory studies. Therefore, procedures were developed for the simultaneous purification of eIF-2, eIF-2B and eIF-2 alpha kinase from kilogram quantities of fresh bovine liver. Briefly, the 0-45% ammonium sulfate precipitate of the 200,000 x g supernatant was solubilized and chromatographed on DEAE-cellulose, heparin-agarose, Mono Q, Mono S, and Superose columns. The availability of purified quantities of these factors will be useful for investigations of molecular mechanisms of action and antibody production.
真核生物起始因子2和2B（eIF-2；eIF-2B）是真核生物蛋白质合成起始限速步骤的组成部分，并参与该过程的调控。当eIF-2的α亚基被eIF-2α激酶磷酸化时，磷酸化的eIF-2α（eIF-2α(P)）会紧密结合eIF-2B，并阻止eIF-2.GDP再循环为蛋白质合成持续起始所需的eIF-2.GTP。这些蛋白质在大鼠肝脏和肌肉细胞质中含量极少，且与数百种其他蛋白质共存，这阻碍了纯化工作以及结构功能和调控研究。因此，开发了从数千克新鲜牛肝中同时纯化eIF-2、eIF-2B和eIF-2α激酶的方法。简要来说，将200,000×g上清液的0-45%硫酸铵沉淀物溶解，并在DEAE-纤维素、肝素琼脂糖、Mono Q、Mono S和Superose柱上进行层析。获得这些纯化因子将有助于研究其分子作用机制和抗体生产。
