Methionyl-tRNA-Met-f deacylase. Purification, characterization, and effects on translational initiation complexes

A methionyl-tRNA-Met-f deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE-cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80,000, which consists of two 40,000-Mr subunits. The mechanism of the Met-tRNA-Met-f deacylase is shown to involve end-product inhibition by the deacylated form of Met-tRNA-Met-f. The methionyl-tRNA-Met-f deacylase is rather specific for Met-tRNA-Met-f as opposed to Met-tRNA-Met-m, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40-S and 60-S subunits inhibit the enzyme, possibly by binding to it. The stability of translational initiation complexes, containing methionyl-tRNA-Met-f, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40-S-subunit . Met-tRNA-Met-f complex was stable in the presence of enzyme. The 80-S complex formed with A-U-G trinucleotide as the message molecule was broken down, whereas the 80-S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met-tRNA-Met-f.