Chevesich J, Chaudhuri J, Maitra U
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461-1602.
J Biol Chem. 1993 Sep 25;268(27):20659-67.
Eukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40 S initiation complex (40 S.mRNA.Met-tRNA(f).eIF-2.GTP). Using assays that measure (a) the release of 32Pi from [gamma-32P]GTP bound to the 40 S initiation complex and (b) the eIF-5-dependent formation of an 80 S initiation complex from a preformed 40 S initiation complex containing bound [35S]Met-tRNA(f), we devised a novel and rapid procedure for purifying eIF-5 from rabbit reticulocyte lysates in high yield. Highly purified eIF-5 is a monomeric protein with a M(r) of about 58,000. However, in partially purified preparations, 58-kDa eIF-5 is associated with other cellular protein(s) and sediments in glycerol gradient centrifugation as a protein of M(r) approximately 160,000. Chicken antibodies to native eIF-5 were isolated from egg yolks of laying hens immunized with rabbit reticulocyte eIF-5. 35S-Labeled eIF-5, isolated from rat anterior pituitary GH3 cells using affinity-purified anti-eIF-5 antibodies, also has an apparent M(r) of 58,000. eIF-5 immunoprecipitated from extracts of 32P-labeled GH3 cells was phosphorylated on serine residues. Phosphopeptide mapping revealed two major sites of phosphorylation, which are distinct from rabbit reticulocyte eIF-5 sites phosphorylated in vitro by casein kinase II. These results demonstrate that eIF-5 is a phosphoprotein that is present in cells as a single molecular form of apparent molecular weight 58,000.
真核生物翻译起始因子5（eIF - 5）促进与40S起始复合物（40S.mRNA.Met - tRNA(f).eIF - 2.GTP）结合的GTP的水解。我们利用检测方法（a）测量从与40S起始复合物结合的[γ - 32P]GTP中释放32Pi以及（b）从含有结合的[35S]Met - tRNA(f)的预先形成的40S起始复合物中依赖eIF - 5形成80S起始复合物，设计了一种从兔网织红细胞裂解物中高产率纯化eIF - 5的新颖快速方法。高度纯化的eIF - 5是一种单体蛋白，分子量约为58,000。然而，在部分纯化的制剂中，58 kDa的eIF - 5与其他细胞蛋白相关联，并且在甘油梯度离心中作为分子量约为160,000的蛋白沉降。从用兔网织红细胞eIF - 5免疫的产蛋母鸡的蛋黄中分离出针对天然eIF - 5的鸡抗体。使用亲和纯化的抗eIF - 5抗体从大鼠垂体前叶GH3细胞中分离出的35S标记的eIF - 5，其表观分子量也为58,000。从32P标记的GH3细胞提取物中免疫沉淀的eIF - 5在丝氨酸残基上被磷酸化。磷酸肽图谱分析揭示了两个主要的磷酸化位点，这与酪蛋白激酶II在体外磷酸化的兔网织红细胞eIF - 5位点不同。这些结果表明，eIF - 5是一种磷蛋白，以表观分子量58,000的单一分子形式存在于细胞中。
