A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from thermus thermophilus HB8

Saho Kamada; Takahiro Okugochi; Kaori Asano; Ryuta Tobe; Hisaaki Mihara; Michiko Nemoto; Kenji Inagaki; Takashi Tamura

doi:10.1080/09168451.2016.1200458

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from thermus thermophilus HB8

Saho Kamada, Takahiro Okugochi, Kaori Asano, Ryuta Tobe, Hisaaki Mihara, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Research output: Contribution to journal › Article › peer-review

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 K_M and k_cat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min-¹ , respectively.

Original language	English
Pages (from-to)	1970-1972
Number of pages	3
Journal	Bioscience, Biotechnology and Biochemistry
Volume	80
Issue number	10
DOIs	https://doi.org/10.1080/09168451.2016.1200458
Publication status	Published - 2016

Keywords

Monoselenophosphate
Non-RI method
Pyruvate pyrophosphate dikinase
Selenophosphate synthetase
Thermus thermophilus hb8

ASJC Scopus subject areas

Biotechnology
Analytical Chemistry
Biochemistry
Applied Microbiology and Biotechnology
Molecular Biology
Organic Chemistry

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10.1080/09168451.2016.1200458

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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.",

keywords = "Monoselenophosphate, Non-RI method, Pyruvate pyrophosphate dikinase, Selenophosphate synthetase, Thermus thermophilus hb8",

author = "Saho Kamada and Takahiro Okugochi and Kaori Asano and Ryuta Tobe and Hisaaki Mihara and Michiko Nemoto and Kenji Inagaki and Takashi Tamura",

note = "Funding Information: This work was supported by JSPS [grant number 15K1469507]; Scientific Research [grant number 15K14695 to T.T]. Publisher Copyright: {\textcopyright} 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry.",

year = "2016",

doi = "10.1080/09168451.2016.1200458",

language = "English",

volume = "80",

pages = "1970--1972",

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issn = "0916-8451",

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T1 - A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from thermus thermophilus HB8

AU - Kamada, Saho

AU - Okugochi, Takahiro

AU - Asano, Kaori

AU - Tobe, Ryuta

AU - Mihara, Hisaaki

AU - Nemoto, Michiko

AU - Inagaki, Kenji

AU - Tamura, Takashi

N1 - Funding Information: This work was supported by JSPS [grant number 15K1469507]; Scientific Research [grant number 15K14695 to T.T]. Publisher Copyright: © 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry.

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

KW - Monoselenophosphate

KW - Non-RI method

KW - Pyruvate pyrophosphate dikinase

KW - Selenophosphate synthetase

KW - Thermus thermophilus hb8

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DO - 10.1080/09168451.2016.1200458

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AN - SCOPUS:84986252902

SN - 0916-8451

VL - 80

SP - 1970

EP - 1972

JO - Bioscience, Biotechnology and Biochemistry

JF - Bioscience, Biotechnology and Biochemistry

IS - 10

ER -

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from thermus thermophilus HB8

Abstract

Keywords

ASJC Scopus subject areas

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