Baan R A, Keller P B, Dahlberg A E
J Biol Chem. 1981 Feb 10;256(3):1063-6.
Protein synthesis initiation factor eIF-2 has been isolated from the high speed supernatant fraction of the yeast Saccharomyces cerevisiae. This purification steps include ammonium sulfate fractionation, column chromatography on Sephacryl 300 and hydroxyapatite, and glycerol gradient sedimentation. Electrophoresis of the purified factor on sodium dodecyl sulfate polyacrylamide gels reveals three polypeptides with a total Mr of 127,000. The molecular weights of the subunits are 31,000, 46,500, and 49,600. The pI of each of these subunits is 4.5, 7.3, and 8.6, respectively. The stoichiometry of the subunits varies from 1:1:1 to 1:0.25:1 suggesting that the active factor may be composed of only two subunits with total Mr of 80,000. The factor is part of a high molecular weight complex during the first steps of the purification. Prior to chromatography on Sephacryl, this complex is broken up in high salt. The activity of the factor is stabilized by inclusion of GDP in all buffers during the preparation and is stimulated by magnesium ion.
蛋白质合成起始因子eIF-2已从酿酒酵母的高速上清液组分中分离出来。该纯化步骤包括硫酸铵分级分离、Sephacryl 300和羟基磷灰石柱色谱以及甘油梯度沉降。纯化后的因子在十二烷基硫酸钠聚丙烯酰胺凝胶上进行电泳，显示出三种多肽，总分子量为127,000。亚基的分子量分别为31,000、46,500和49,600。这些亚基的pI分别为4.5、7.3和8.6。亚基的化学计量比从1:1:1到1:0.25:1不等，这表明活性因子可能仅由两个总分子量为80,000的亚基组成。在纯化的第一步中，该因子是高分子量复合物的一部分。在Sephacryl柱色谱之前，这种复合物在高盐条件下被分解。在制备过程中，通过在所有缓冲液中加入GDP可稳定该因子的活性，并且镁离子可刺激其活性。
