Panniers R, Stewart E B, Merrick W C, Henshaw E C
J Biol Chem. 1985 Aug 15;260(17):9648-53.
Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.
几乎所有被研究的生物体在温度升高时都会对整体蛋白质合成产生显著抑制，但会增加一组特定蛋白质（即所谓的热休克蛋白）的合成。我们从热休克的艾氏腹水癌细胞中制备了一种无细胞蛋白质合成系统（裂解物），该系统反映了完整细胞在高温下蛋白质合成的抑制情况。我们从艾氏细胞中分离并部分纯化了热休克细胞裂解物的一种刺激物。经过四个纯化步骤，该刺激物在色谱上与真核起始因子4F（eIF - 4F）相同，eIF - 4F是一种特异性结合mRNA帽结构的起始因子。因此，我们测试了高度纯化的网织红细胞eIF - 4F对热休克细胞裂解物的影响。添加高度纯化的eIF - 4F可强烈刺激蛋白质合成。在热休克裂解物中，高（70 mM）氯化钾浓度下的合成比低浓度下更受抑制，并且在较高氯化钾浓度下eIF - 4F的刺激作用相应更大，以至于在所有氯化钾浓度下蛋白质合成速率都恢复到对照（非热休克裂解物）水平。此外，在70 mM氯化钾条件下，在热休克裂解物中，68 kDa热休克蛋白的合成比大多数非热休克蛋白的合成受抑制程度小得多，并且eIF - 4F对68 kDa蛋白合成的刺激程度比非热休克蛋白小得多。因此，添加纯化的eIF - 4F可逆转高温对裂解物中所反映的艾氏细胞的影响。所以，我们提出热休克的艾氏细胞中翻译的抑制是eIF - 4F功能失活的结果。
