Isolation and characterization of two forms of Met-tRNAf deacylase from rabbit reticulocyte ribosomes.
We have separated and purified two forms of Met-tRNAf deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes. Deacylase I is a minor activity with an S20,w of 5.9, D20,w of 4.9 and Mr of 110 000, while deacylase II is the major activity with an S20,w of 3.3, D20,w of 7.1 and Mr of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [35S]Met-tRNAf to [35S]methionine and tRNAfMet and have no effect on reticulocyte [35S]fMet-tRNAf, [3H]Ala-tRNA or [3H]Lys-tRNA. However, while deacylase I has similar activity throughout the pH range of 6.1-8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1. In addition, deacylase II shows a much greater affinity for pure Met-tRNAf than deacylase I (Km of 1.5-3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNAfMet, deacylase I is inhibited similarly by any added tRNA.
D000217:Acyltransferases / Q000302:isolation & purification*; D019881:Aminoacyltransferases; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D012270:Ribosomes / Q000201:enzymology*
D007527:Isoenzymes; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D000217:Acyltransferases; D019881:Aminoacyltransferases; C021560:methionyl-tRNA-Met-f deacylase