Lamphear B J, Panniers R
Cancer Center, University of Rochester, New York 14642.
J Biol Chem. 1991 Feb 15;266(5):2789-94.
Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Recently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:1 molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat-shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine triphosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5' mRNA cap. We have ruled out proteolysis and competitive inhibitors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non-competitive inhibitor blocks complex binding to cap. We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, p220.p28 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.
由热休克艾氏腹水癌细胞制备的无细胞蛋白质合成系统保留了在细胞水平上观察到的翻译抑制作用。最近，我们发现一种高度纯化的帽结合蛋白复合物，由真核起始因子4F的p220和p28亚基以1:1的摩尔比组成，可在这些无细胞翻译系统中恢复蛋白质合成（Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333 - 5336）。在此，我们估计了细胞提取物中能够恢复热休克细胞蛋白质合成的帽结合复合物的量。我们发现热休克细胞提取物中的恢复活性降低。此外，通过7 - 甲基鸟苷三磷酸琼脂糖亲和层析从热休克细胞提取物中纯化得到的帽结合复合物较少，我们得出结论，热休克会损害复合物与5' mRNA帽的结合。我们排除了蛋白水解和竞争性抑制剂作为这种损害的介导因素。然而，我们无法区分两种可能的解释：（i）p220与p28的结合减少，或（ii）一种非竞争性抑制剂阻断复合物与帽的结合。我们还研究了热休克对两种形式的p28、p220·p28复合物和不含p220的p28的磷酸化状态的影响。热休克期间，这两种形式的磷酸化水平均降低。文中讨论了这些变化的意义。
