Shalak V F, Budkevich T V, Negrutskił B S, El'skaia A V
Ukr Biokhim Zh (1978). 1997 Mar-Apr;69(2):104-9.
Fast, efficient and gentle method of elongation factor 1 alpha (EF-1 alpha) purification from rabbit liver has been developed. Isolation procedure consists of three steps: gel-filtration of postmithohondrial supernatant on Sephacryl S-400, anion(DEAE-Cellulose)- and cation(SP-Sepharose)- exchange chromatographies. The procedure takes two-three days. Minimal number of the purification steps and the only one buffer usage during entire purification provide the high speed and efficiency of the method. Purity of EF-1 alpha preparation obtained was more than 90% as judged from DS-Na polyacrylamide gel electrophoresis. EF-1 alpha was demonstrated to be highly active in three different assays: the [3H]GDP-binding, the stimulation of [14C]phenylalanyl-tRNA binding to poly(U)-programmed 80S ribosomes and the stimulation of poly[14C]phenylalanine synthesis on poly(U)-programmed 80S ribosomes in the presence of EF-2. Since EF-1 alpha may exist in a cell as either GDP- or GTP-bound form the nature of nucleotide bound was studied using high pressure liquid chromatography (HPLC) analysis. Each molecule of EF-1 alpha appears to contain one molecule of GDP.
已开发出一种从兔肝中快速、高效且温和地纯化延伸因子1α（EF-1α）的方法。分离过程包括三个步骤：线粒体后上清液在Sephacryl S-400上进行凝胶过滤、阴离子（DEAE-纤维素）和阳离子（SP-琼脂糖）交换色谱。该过程需要两到三天。纯化步骤数量最少且在整个纯化过程中仅使用一种缓冲液，这使得该方法具有高速和高效性。根据十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断，获得的EF-1α制剂纯度超过90%。EF-1α在三种不同的测定中显示出高活性：[3H]GDP结合、刺激[14C]苯丙氨酰-tRNA与聚（U）编程的80S核糖体结合以及在EF-2存在下刺激聚（U）编程的80S核糖体上的聚[14C]苯丙氨酸合成。由于EF-1α在细胞中可能以结合GDP或GTP的形式存在，因此使用高压液相色谱（HPLC）分析研究了结合核苷酸的性质。每个EF-1α分子似乎含有一个GDP分子。
