Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.

Abstract

To define the regulation mechanism of protein kinase that is activated in heme deficiency and that inhibits initiation or protein synthesis, the heme-reversible form of the protein kinase was isolated and purified from rabbit reticulocytes. The inhibitory activity was in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrated as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor became self-phosphorylated in the presence of ATP. The phosphorylated protein and inhibitory activity copurified. The inhibitor produced characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylated the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). The inhibition was reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor was no longer inhibitory after incubation with 20 .mu.M hemin. Incubation with hemin inhibited self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiated the inhibition of protein synthesis in the subsequent incubation, as did treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appeared related. Hemin apparently acts directly on the heme-reversible inhibitor.