Isolation of a heme-controlled inhibitor of translation that blocks the interaction between messenger rna and eukaryotic initiation factor 2.
A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose. The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of alpha-over beta-globin synthesis. The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 degrees C, pH 2 or pH 12. The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein. Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity. The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNAfMet, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA. In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA. The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D015852:Eukaryotic Initiation Factor-2; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects*; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D012333:RNA, Messenger; D006418:Heme; D011494:Protein Kinases; D019892:eIF-2 Kinase