Fate of reversing factor during restoration of protein synthesis by hemin or GTP in heme-deficient reticulocyte lysates.
Fate of reversing factor during restoration of protein synthesis by hemin or GTP in heme-deficient reticulocyte lysates.
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血红素缺乏的网织红细胞裂解液中血红素或 GTP 恢复蛋白质合成过程中逆转因子的命运。
DOI：
10.1073/pnas.83.5.1217
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发表时间：
1986
影响因子：
11.1
通讯作者：
London,IM
中科院分区：
文献类型：
--
作者：
Matts,RL;Levin,DH;London,IM
血红素缺乏的网织红细胞裂解物中蛋白质合成的抑制可通过加入氯化血红素（20 μ M）或MgGTP（2 mM）逆转。回收率是快速的，并且在添加任一组分后的几分钟内接近对照动力学。蛋白质合成的恢复依赖于功能逆转因子（RF）的可用性。在回收期间，通过使用在生理条件下测量裂解物中RF活性的方法监测RF的去向。在完全抑制的裂解物中，RF被隔离在不可解离的15 S [RF。eIF-2（α P）]复合物（其中eIF-2表示真核起始因子2），其中RF活性无功能，无法测定。在通过氯化血红素或MgGTP抑制的裂解物中拯救蛋白质合成的第一步是抑制血红素调节的eIF-2 α激酶，其使得内源性磷酸酶能够使eIF-2（α P）和[RF]去磷酸化。eIF-2（α P）]。约50%的隔离RF活性的释放足以支持最佳的恢复动力学。氯化血红素和MgGTP都通过阻断裂解物中血红素调节的eIF-2 α激酶的活化和/或活性来逆转抑制。MgGTP对eIF-2 α激酶发挥其作用的结论得到几个体外发现的支持：（i）2 mM MgGTP抑制纯化的血红素调节的eIF-2 α激酶的自磷酸化并消除其磷酸化eIF-2 α的能力;（ii）2 mM MgGTP不能置换二元复合物中的GDP [eIF-2 . GDP]或[eIF-2（α P）。国内生产总值]的质量行动;和（三）RF在[RF。eIF-2（α P）]复合物不被2 mM MgGTP解离。
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.