Regulation of protein synthesis in reticulocyte lysates. Characterization of the inhibitor generated in the postribosomal supernatant by heating at 44 degrees C.
Heating of a rabbit reticulocyte lysate at 44 degrees C, in the presence of optimal concentrations of haemin, results in an inhibition of protein synthesis and in the appearance of an inhibitory activity in the postribosomal supernatant. These effects of supraoptimal heating are similar to those observed in lysates and supernatants incubated at physiological temperatures in the absence of haemin. In this paper we examined whether the haem-regulated inhibitor produced by treating postribosomal supernatant with N-ethylmaleimide and the inhibitor generated by heating at 44 degrees C in the presence of haemin are the same molecular entity. Both inhibitors behaved similarly through a partial purification consisting of 50% (NH4)2SO4 precipitation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography, and had the same pattern of polypeptides after polyacrylamide gel electrophoresis. However, when incubated with an antiserum to the haem-regulated inhibitor the activity of the 44 degrees C heated supernatant was not neutralized, whereas that of its more purified fractions was. This apparent contradiction was shown to be due to an interference of the immune serum assay by the levels of proinhibitor and haemoglobin present in the crude supernatant. Further experiments, with extensively diluted 44 degrees C supernatants or with isolated proinhibitor subsequently heated, are consistent with the conclusion that both heating at supraoptimal temperatures and incubating in the absence of haem finally activate the same inhibitor.
D000818:Animals; D002474:Cell-Free System / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006358:Hot Temperature; D007158:Immunologic Techniques; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors*; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000494:pharmacology*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D010448:Peptide Initiation Factors; D006427:Hemin; D011494:Protein Kinases; D005033:Ethylmaleimide