Wu S, Gupta S, Chatterjee N, Hileman R E, Kinzy T G, Denslow N D, Merrick W C, Chakrabarti D, Osterman J C, Gupta N K
Department of Chemistry, University of Nebraska, Lincoln 68588.
J Biol Chem. 1993 May 25;268(15):10796-801.
The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprotein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-stranded RNA-activated inhibitor. This promotes protein synthesis in the presence of eIF-2 kinases present in animal cells (Ray, M. K., Datta, B., Chakraborty, A., Chattopadhyay, A., Meza-Keuthen, S., and Gupta, N. K. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543). In this study, the primary structure of rat p67 is determined by cDNA cloning. Based on the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding p67 cDNA fragment from rat liver first strand cDNA. The amplified DNA was then used as a probe to screen a rat tumor hepatoma (KRC-7) cDNA library, and a positive clone covering the entire coding region was obtained. From the cDNA sequence, an open reading frame that encodes p67 as a 480-amino acid protein with a molecular mass of 53 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled translation in micrococcal nuclease-treated reticulocyte lysate. The translated product migrated similarly to p67 in SDS-polyacrylamide gel electrophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (approximately 2 kilobases) of mRNA. The deduced amino acid sequence of the protein showed a highly charged N-terminal region composed of two basic polylysine blocks and an acidic aspartic acid block. The protein also exhibits significant sequence identity in the N-terminal region with human eIF-2 beta-subunit.
真核生物起始因子2（eIF-2）相关的67 kDa糖蛋白（p67）可保护eIF-2α亚基免受eIF-2激酶（如血红素调节抑制剂和双链RNA激活抑制剂）的抑制性磷酸化。这在动物细胞中存在eIF-2激酶的情况下促进蛋白质合成（Ray，M.K.，Datta，B.，Chakraborty，A.，Chattopadhyay，A.，Meza-Keuthen，S.和Gupta，N.K.（1992年）美国国家科学院院刊89，539 - 543）。在本研究中，通过cDNA克隆确定了大鼠p67的一级结构。基于胰蛋白酶和溴化氰切割的重叠片段的部分氨基酸序列，合成了简并寡核苷酸，并用作聚合酶链反应的引物，以从大鼠肝脏第一链cDNA中扩增相应的p67 cDNA片段。然后将扩增的DNA用作探针筛选大鼠肿瘤肝癌（KRC - 7）cDNA文库，并获得了覆盖整个编码区的阳性克隆。根据cDNA序列，预测未糖基化的蛋白质的开放阅读框编码一个480个氨基酸的蛋白质p67，分子量为53千道尔顿。通过在微球菌核酸酶处理的网织红细胞裂解物中的体外转录偶联翻译进一步表征克隆的cDNA。翻译产物在SDS - 聚丙烯酰胺凝胶电泳中的迁移与p67相似，并且被抗p67抗体沉淀。大鼠肝脏聚腺苷酸加尾RNA的Northern印迹分析显示单一大小类别的mRNA（约2千碱基）。该蛋白质的推导氨基酸序列显示出由两个碱性多聚赖氨酸块和一个酸性天冬氨酸块组成的高度带电的N端区域。该蛋白质在N端区域与人eIF-2β亚基也表现出显著的序列同一性。
