title: A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8.
abstract: Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min(-1), respectively.
authors.affiliation: a Graduate School of Life and Environmental Science , Okayama University , Okayama , Japan.
authors.forename: Saho
authors.initials: S
authors.lastname: Kamada
authors.affiliation: a Graduate School of Life and Environmental Science , Okayama University , Okayama , Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Okugochi
authors.affiliation: a Graduate School of Life and Environmental Science , Okayama University , Okayama , Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Asano
authors.affiliation: b College of Life Sciences , Ritsumeikan University , Kusatsu , Japan.
authors.forename: Ryuta
authors.initials: R
authors.lastname: Tobe
authors.affiliation: b College of Life Sciences , Ritsumeikan University , Kusatsu , Japan.
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Mihara
authors.affiliation: a Graduate School of Life and Environmental Science , Okayama University , Okayama , Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Nemoto
authors.affiliation: a Graduate School of Life and Environmental Science , Okayama University , Okayama , Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
authors.affiliation: a Graduate School of Life and Environmental Science , Okayama University , Okayama , Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tamura
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D010770:Phosphotransferases / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D019289:Pyruvic Acid; D010770:Phosphotransferases; C062762:selenophosphate synthetase
keywords: Thermus thermophilus HB8; monoselenophosphate; non-RI method; pyruvate pyrophosphate dikinase; selenophosphate synthetase