title: Mode of action of the heme-controlled translational inhibitor: relationship of eukaryotic initiation factor 2-stimulating protein to translation restoring factor.
abstract: We have purified the translation restoring factor (RF) and the eukaryotic initiation factor 2 (eIF-2) stimulating protein (ESP) to near homogeneity from the postribosomal supernatant and the ribosomal salt wash, respectively, of rabbit reticulocyte lysate. They were isolated in the form of eIF-2 complexes, apparently in a 1:1 ratio. Their virtually identical NaDodSO4/polyacrylamide gel electrophoretic patterns show, in addition to the eIF-2 alpha (38,000), beta (52,000), and gamma (54,000) bands, peptide bands at approximately 80, 65, 57, 40, and 32 kilodaltons. The apparent Mr of either complex is about 450,000, whereas that of free translation restoring factor (RF) is approximately 25,000. At 0.5 mM Mg2+, both ESP and RF stimulate ternary complex (eIF-2.GTP.Met-tRNAi) formation catalytically with unphosphorylated eIF-2. Phosphorylation of the eIF-2 alpha subunit by preincubation with eIF-2 alpha kinase and ATP, which virtually blocks eIF-2-ESP interaction, results in only partial blocking of the interaction with RF. This may explain the translation restoring activity of RF.
mesh_terms: D000818:Animals; D015852:Eukaryotic Initiation Factor-2; D005786:Gene Expression Regulation; D005914:Globins / Q000096:biosynthesis; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000302:isolation & purification* / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D019892:eIF-2 Kinase
chemical_list: D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; C025207:eIF-2 Met-tRNA(f)(met) GDPCP; D006418:Heme; D006427:Hemin; D006160:Guanosine Triphosphate; D005914:Globins; D012343:RNA, Transfer; D011494:Protein Kinases; D019892:eIF-2 Kinase