Some evidence of the enzymatic conversion of bovine suppressor phosphoseryl-tRNA to selenocysteyl-tRNA

In order to clarify the mechanisms of selenocysteine incorporation into glutathione peroxidase, some evidence to show the in vitro conversion of phosphoseryl-tRNA to selenocysteyl-tRNA is reported. [³H]Phosphoseryl-tRNA was incubated in a reaction mixture composed of SeO₂, glutathione and NADPH in the presence of selenium-transferase partially purified. Analyses of amino acids on the product tRNA showed that a part (4%) of [³H]phosphoseryl-tRNA was changed to [³H]selenocysteyl-tRNA. The conversion from seryl-tRNA^su or major seryl-tRNA_IGA was not found. Selenium-transferase was essential for the conversion. [³H]Selenocysteine, liberated from the tRNA, was modified with iodoacetic acid. The product was confirmed to be carboxymethyl-selenocysteine by two-dimensional TLC. Selenocysteyl-tRNA^su should be used to synthesize glutathione peroxidase by co-translational mechanisms.     

Purification and properties of suppressor seryl-tRNA: ATP phosphotransferase from bovine liver

Seryl-tRNASerCmCA: ATP phosphotransferase was purified 1200-fold from bovine liver by ultracentrifugation at 150 000 X g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, chromatography on hydroxyapatite, gel filtration on Sephacryl S-300 and affinity chromatography on Blue Sepharose. Molecular mass was estimated as 135-145 kDa. The Km values for ATP and ser-tRNASerCmCA were 2 mM and 21 nM, respectively. This enzyme did not react with ser-tRNASerIGA, tyr-tRNA or thr-tRNA.