title: Some properties of murine selenocysteine synthase.
abstract: Selenocysteine (Scy) was synthesized on natural opal suppressor tRNA(Ser) by conversion from seryl-tRNA. We studied the mechanisms of the synthesis of mammalian Scy-tRNA using hydro[75Se]selenide (H75Se-). We found Scy synthase activity in the 105,000 g supernatant of a murine liver extract. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at 0.12 M-KCl. The reaction mixture for synthesis of Scy-tRNA contained suppressor tRNA, serine, ATP, seryl-tRNA synthetase (SerRS), HSe- and the enzyme to synthesize Scy-tRNA. These are all essential for the synthesis of Scy-tRNA. Scy in the tRNA product was confirmed by five t.l.c. systems. The conversion from seryl-tRNA to Scy-tRNA was also confirmed with the use of [14C]- and [3H]-serine. The apparent Km values for the substrates serine, tRNA, ATP and HSe- were 30 microM, 140 nM, 2 mM and 40 nM respectively. The active eluates from DEAE-cellulose contained no tRNA kinase. This result showed that Scy-tRNA was not synthesized through phosphoseryl-tRNA. ATP was necessary when Scy-tRNA was synthesized from seryl-tRNA and HSe-. Therefore ATP is used for not only the synthesis of seryl-tRNA but also for the synthesis of Scy-tRNA from seryl-tRNA. The active fraction from DEAE-cellulose was chromatographed on Sephacryl S-300, but the activity disappeared. However, the activity was recovered by mixing the eluates corresponding to proteins of 500 kDa and 20 kDa. In order to examine the binding of HSe- to proteins, a mixture of the active fraction, H75Se- and ATP was analysed by chromatography on Sephacryl S-300. The 75Se radioactivity was found at the position of a 20 kDa protein in the presence of ATP. Thus the 20 kDa protein plays a role in binding HSe- in the presence of ATP. The 500 kDa protein must have a role in the synthesis of Scy-tRNA. There are two natural suppressor serine tRNAs, tRNA(NCA) and tRNA(CmCA), in cell cytosol. The present paper shows that the suppressor tRNA fraction, eluted later on benzoylated DEAE-(BD-)cellulose, is a better substrate with which to synthesize Scy-tRNA. Thus we consider that murine Scy-tRNA is synthesized from a suppressor seryl-tRNA on the 500 kDa protein with the activated HSe-, which is synthesized with ATP on the 20 kDa protein. This mammalian mechanism used to synthesize Scy is similar to that seen in Escherichia coli.
authors.affiliation: Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Mizutani
authors.forename: H
authors.initials: H
authors.lastname: Kurata
authors.forename: K
authors.initials: K
authors.lastname: Yamada
authors.forename: T
authors.initials: T
authors.lastname: Totsuka
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008813:Mice, Inbred ICR; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis / Q000235:genetics; D012362:RNA, Transfer, Ser / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D018036:Selenium Compounds; D012644:Selenium Radioisotopes; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D012346:RNA, Transfer, Amino Acyl; D012362:RNA, Transfer, Ser; D018036:Selenium Compounds; D012644:Selenium Radioisotopes; C112705:selenocysteinyl-tRNA; D014166:Transferases; C060600:selenium transferase; D012643:Selenium; C026372:hydrogen selenide