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Mechanism of stimulation of globin synthesis by cyclic amp and gtp in a rabbit reticulocyte lysate system


, : Mechanism of stimulation of globin synthesis by cyclic amp and gtp in a rabbit reticulocyte lysate system. Journal of Biochemistry (Tokyo) 97(5): 1289-1300

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. [.alpha. subunit of eukaryotic initiation factor .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 the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and cAMP and GTP inhibited the phosphorylation of eIF-2.alpha. in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, 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) [eIF-2 phosphorylated in eIF-2.alpha.]. The rates of the restored synthesis of globin were lower than the control. cAMP promoted the release of [3H]GDP from the eIF-2(.alpha.P).cntdot.[3H]GDP complex and the formation of eIF-2(.alpha.P).cntdot.eIF-2B complex. Steps involved in the restorative effect of these compounds evidently may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. Data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. 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).cntdot.eIF-2B complex. GTP prevented 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.

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