文章基本信息

标题：Site-specific phosphorylation of the α subunit of eukaryotic initiation factor eIF-2 by the heme-regulated and double-stranded RNA-activated eIF-2α kinases from rabbit reticulocyte lysates
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作者：Vivian Ernst ; Daniel H. Levin ; Alena Leroux 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1980
卷号：77
期号：3
页码：1286-1290
DOI：10.1073/pnas.77.3.1286
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The site specificity of phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF-2) by the heme-regulated and double-stranded RNA-activated eIF-2 kinases were compared by phosphopeptide mapping. eIF-2 was maximally phosphorylated in vitro with [{gamma}-32P]ATP and either crude or partially purified preparations of the kinases. 32P-Labeled eIF-2 was isolated by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The fixed, stained, and dried polypeptide band was excised and then exhaustively digested directly in the gel slice with one of several proteases (trypsin, chymotrypsin, subtilisin, or thermolysin); the resultant [32P]phosphopeptides were analyzed by one-dimensional chromatography or by two-dimensional chromatography and high-voltage electrophoresis. In addition, limited proteolysis of [32P]eIF-2 contained in fixed, dried, and stained gel slices was achieved with Staphylococcus aureus protease V8, chymotrypsin, or subtilisin, and the partial 32P-labeled cleavage products were analyzed by gel electrophoresis. Each protease produced distinct and reproducible [32P]phosphopeptide profiles after partial or exhaustive proteolysis of [32P]eIF-2. With a given protease, identical [32P]phosphopeptide patterns were obtained whether eIF-2 was phosphorylated by the heme-regulated or the double-stranded RNA-activated kinase. These data indicate that, in vitro, the kinases phosphorylate sites on eIF-2 that are identical or proximally located in the primary sequence. In this report we also provide preliminary evidence that the two eIF-2 kinases activated in lysates by heme deficiency or double-stranded RNA phosphorylate site(s) of endogenous eIF-2 that are similar, if not identical, to the sites phosphorylated in vitro with partially purified eIF-2 kinase(s) and eIF-2.
关键词：protein synthesis regulation ; proteolytic digestion ; [³²P]phosphopeptide analysis