Suzuki H, Mukouyama E B
J Biochem. 1985 May;97(5):1289-300. doi: 10.1093/oxfordjournals.jbchem.a135180.
The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.
在血红素缺乏的兔网织红细胞裂解物中，珠蛋白合成的抑制是由于一种血红素控制的翻译抑制剂（HCI）的激活，该抑制剂特异性地使真核起始因子2α（eIF-2α）磷酸化。当在孵育初期加入高浓度的环磷酸腺苷（cAMP，5 - 10 mM）和鸟苷三磷酸（GTP，1 - 2 mM）时，它们能刺激血红素缺乏的裂解物中的珠蛋白合成。利用血红素缺乏的裂解物、经N - 乙基马来酰胺（NEM）处理并补充了HCI的裂解物以及部分纯化的起始因子eIF-2，研究了cAMP和GTP的刺激机制。由于这些化合物对珠蛋白合成的刺激必定是由于防止了珠蛋白合成的抑制，或由于恢复了珠蛋白合成，或两者兼而有之，因此研究了这些化合物的预防和恢复作用。关于预防作用，观察到：a）网织红细胞核糖体后上清液中HCI的激活被GTP阻止，但未被cAMP阻止；b）cAMP和GTP抑制血红素缺乏的裂解物中eIF-2α的磷酸化。关于cAMP和GTP的恢复作用，观察到：c）这些化合物恢复了珠蛋白合成以及[35S]甲硫氨酰 - tRNAf与40S核糖体亚基的结合，并促进了eIF-2（αP）的去磷酸化；d）恢复的珠蛋白合成速率低于对照；e）cAMP促进了[3H]GDP从eIF-2（αP）·[3H]GDP复合物中的释放以及eIF-2（αP）·eIF-2B复合物的形成。发现（d）表明这些化合物恢复作用所涉及的步骤可能对血红素缺乏的裂解物中珠蛋白合成的刺激没有贡献。关于cAMP和GTP预防和恢复作用的数据表明，这些化合物影响多个步骤。也就是说，cAMP抑制eIF-2α的磷酸化，并促进GDP从eIF-2中的释放以及eIF-2（αP）·eIF-2B复合物的形成，而GTP既阻止HCI的激活，也阻止eIF-2α的磷酸化。尽管cAMP和GTP影响多个步骤，但提示cAMP通过抑制eIF-2α的磷酸化来刺激珠蛋白合成，而GTP主要通过防止血红素缺乏的裂解物中HCI的激活来刺激珠蛋白合成。
