Chakravarti D, Maiti T, Maitra U
Department of Developmental Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461.
J Biol Chem. 1993 Mar 15;268(8):5754-62.
Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates. The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000. However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed. Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other. In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000. Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates. Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation. When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed. No precursor forms of molecular weight higher than 56,000 were detected in any preparations. These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.
真核生物翻译起始因子5（eIF - 5）可催化与40S核糖体起始复合物结合的GTP水解，已从酵母细胞裂解物中纯化出来。从凝胶过滤柱上洗脱下来的纯化因子是一种表观分子量（M(r)）为45,000 - 50,000的蛋白质。然而，当在十二烷基硫酸钠存在的情况下通过聚丙烯酰胺凝胶电泳分析纯化制剂时，观察到两种表观分子量分别为54,000和56,000的不同多肽。发现这两种多肽各自都具有eIF - 5活性，并且它们在免疫上相互关联。然而，在酵母eIF - 5纯度较低的制剂中，相当一部分eIF - 5活性从凝胶过滤柱上洗脱下来时是一种分子量大于140,000的蛋白质。因此，采用免疫化学方法来确定粗制酵母细胞裂解物中eIF - 5的分子结构。用纯化的酵母eIF - 5在兔体内制备抗血清，并证明其在抑制eIF - 5介导的80S起始复合物形成方面具有高效性。当用亲和纯化的抗eIF - 5抗体进行免疫印迹分析粗制eIF - 5制剂以及直接裂解到含有3%十二烷基硫酸钠的变性缓冲液中的酵母细胞时，观察到一条主要的免疫反应性多肽（表观M(r) = 54,000）和一条次要条带（表观M(r) = 56,000）。在任何制剂中均未检测到分子量高于56,000的前体形式。这些结果表明酵母eIF - 5是一种表观M(r) = 50,000 - 56,000的单体蛋白。
