文章基本信息

标题：Fate of reversing factor during restoration of protein synthesis by hemin or GTP in heme-deficient reticulocyte lysates
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作者：R L Matts ; D H Levin ; I M London 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：1986
卷号：83
期号：5
页码：1217-1221
DOI：10.1073/pnas.83.5.1217
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The inhibition of protein synthesis in hemedeficient reticulocyte lysates is reversed by the addition of hemin (20 microM) or MgGTP (2 mM). The rate of recovery is rapid and approaches control kinetics within a few minutes after the addition of either component. The restoration of protein synthesis is dependent upon the availability of functional reversing factor (RF). The fate of RF was monitored during recovery by using a method that measures RF activity in the lysate under physiological conditions. In the fully inhibited lysate, RF is sequestered in a nondissociable 15S [RF . eIF-2(alpha P)] complex (where eIF-2 indicates eukaryotic initiation factor 2) in which RF activity is not functional and cannot be assayed. The first step in the rescue of protein synthesis in inhibited lysates by hemin or MgGTP is the inhibition of heme-regulated eIF-2 alpha kinase, which enables endogenous phosphatase to dephosphorylate eIF-2(alpha P) and [RF . eIF-2(alpha P)]. The release of approximately 50% of the sequestered RF activity is sufficient to support optimal kinetics of recovery. Hemin and MgGTP both reverse inhibition by blocking the activation and/or activity of heme-regulated eIF-2 alpha kinase in the lysate. The conclusion that MgGTP exerts its effect on eIF-2 alpha kinase is supported by several in vitro findings: (i) 2 mM MgGTP inhibits the autophosphorylation of purified heme-regulated eIF-2 alpha kinase and abolishes its ability to phosphorylate eIF-2 alpha; (ii) 2 mM MgGTP cannot displace GDP in the binary complexes [eIF-2 . GDP] or [eIF-2(alpha P) . GDP] by mass action; and (iii) RF in the [RF . eIF-2(alpha P)] complex is not dissociated by 2 mM MgGTP.