Isolation and characterization of two forms of Met-tRNA f deacylase from rabbit reticulocyte ribosomes

Abstract

We have separated and purified two forms of Met-tRNA f 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 S 20,w of 5.9, D 20,w of 4.9 and M r of 110 000, while deacylase II is the major activity with an S 20,w of 3.3, D 20,w of 7.1 and M r of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [ 35S]Met-tRNA f to [ 35S]methionine and tRNA Met f and have no effect on reticulocyte [ 35S]fMet-tRNA f, [ 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-tRNA f than deacylase I ( K m of 1.5–3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNA Met f, deacylase I is inhibited similarly by any added tRNA.