Study of mammalian selenocysteyl-tRNA synthesis with [75Se]HSe

Abstract

The mechanisms of the synthesis of mammalian selenocysteyl-(Scy)-tRNA were studied using [75SE]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy-tRNA. [75Se]Scy, liberated from aminoacyl-tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy-tRNA synthesis was found in the supernatant at 105,000 x g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl-tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNA(IGA) is not a substrate on which to synthesize Scy-tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy-tRNA preparation on Sephacryl S-200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE-cellulose did not contain tRNA kinase, therefore Scy-tRNA must be directly synthesized from seryl-tRNA, not through phosphoseryl-tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324].