Use of an antibody to characterize and determine the role of the major Met-tRNAf deacylase from rabbit reticulocyte ribosomes.
Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(alpha) results, secondarily, in the enzymatic deacylation of Met-tRNAf on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNAf deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNAf deacylase II and has no effect on Met-tRNAf deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNAf deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte lysate incubated in the absence of hemin or presence of hemin plus 0.1 microgram/ml poly(I X C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNAf and increase the level of Met-tRNAf bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNAf in the 48 S complexes that accumulate.
D000217:Acyltransferases / Q000276:immunology / Q000378:metabolism*; D019881:Aminoacyltransferases; D000818:Animals; D001853:Bone Marrow / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D012270:Ribosomes / Q000201:enzymology*
D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D008715:Methionine; D000217:Acyltransferases; D019881:Aminoacyltransferases; C021560:methionyl-tRNA-Met-f deacylase