Characterization of a rabbit reticulocyte supernatant factor that reverses the translational inhibition of hemin deficiency.
The control of protein synthesis by hemin in rabbit reticulocyte lysate is mediated by the formation of a high molecular weight protein inhibitor (or translational repressor) of polypeptide chain initiation from a precursor (prorepressor), which acts by phosphorylating the Mr 35,000 (alpha) subunit of eIF-2. We originally isolated a post-ribosomal supernatant factor from reticulocyte lysate, distinct from soluble eIF-2, that could completely reverse the inhibition of protein synthesis that occurs when reticulocyte lysate is incubated in the absence of hemin. We have found that this supernatant factor promotes the inactivation of the intermediate form of the translational repressor (generated within 1 h of incubation of the prorepressor in the absence of hemin), but only in the presence of GTP. This inactivation is not seen with the irreversible form of the translational repressor (generated after prolonged incubation of the prorepressor) and does not occur with other nucleoside triphosphates, dGTP or GDP. In addition, the inactivation reaction is not dependent upon Mg2+ and is not mediated by cyclic GMP. When lysate samples were pulsed with [gamma-32P]ATP at 15 min of incubation, the addition of the supernatant factor at zero time or after 7 min was associated with preventing or reversing the phosphorylation of eIF-2 alpha. In contrast, another soluble reticulocyte protein termed reversing factor can be separated from the supernatant factor on phosphocellulose, partly overcomes the effect of the irreversible translational repressor, and is associated with increased phosphorylation of eIF-2 alpha.
D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis; D015852:Eukaryotic Initiation Factor-2; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D019892:eIF-2 Kinase
D001798:Blood Proteins; D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D006418:Heme; D006427:Hemin; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D008274:Magnesium