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

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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 × 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 tRNAIGA 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].