Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.
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.
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*
D010448:Peptide Initiation Factors; D012097:Repressor Proteins; D014157:Transcription Factors; D006418:Heme; D006427:Hemin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D005033:Ethylmaleimide