title: Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.
abstract: To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.
mesh_terms: D000255:Adenosine Triphosphate; D000818:Animals; D004789:Enzyme Activation; D005033:Ethylmaleimide; D006418:Heme / Q000494:pharmacology*; D006427:Hemin / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D012097:Repressor Proteins / Q000302:isolation & purification*; D012156:Reticulocytes / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification*
chemical_list: D010448:Peptide Initiation Factors; D012097:Repressor Proteins; D014157:Transcription Factors; D006418:Heme; D006427:Hemin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D005033:Ethylmaleimide