Fetal mouse selenophosphate synthetase 2 (SPS2)

biological activities of mutant forms in Escherichia coli.

T. S. Kim, M. H. Yu, Y. W. Chung, Jae-Hong Kim, Eui Ju Choi, K. Ahn, Ick Young Kim

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.

Original languageEnglish
Pages (from-to)422-428
Number of pages7
JournalMolecules and Cells
Volume9
Issue number4
Publication statusPublished - 1999 Aug 31

Fingerprint

Escherichia coli
Genes
Selenocysteine
Selenium Compounds
Mutation
Mouse Sephs2 protein
Terminator Codon
COS Cells
Enzymes
Open Reading Frames
Cysteine
Proteins
Embryonic Structures
Complementary DNA
Adenosine Triphosphate
Monoclonal Antibodies
selenophosphate synthetase

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Fetal mouse selenophosphate synthetase 2 (SPS2) : biological activities of mutant forms in Escherichia coli. / Kim, T. S.; Yu, M. H.; Chung, Y. W.; Kim, Jae-Hong; Choi, Eui Ju; Ahn, K.; Kim, Ick Young.

In: Molecules and Cells, Vol. 9, No. 4, 31.08.1999, p. 422-428.

Research output: Contribution to journalArticle

@article{141cdb3872b840dbad5f601fbd157090,
title = "Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli.",
abstract = "A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.",
author = "Kim, {T. S.} and Yu, {M. H.} and Chung, {Y. W.} and Jae-Hong Kim and Choi, {Eui Ju} and K. Ahn and Kim, {Ick Young}",
year = "1999",
month = "8",
day = "31",
language = "English",
volume = "9",
pages = "422--428",
journal = "Molecules and Cells",
issn = "1016-8478",
publisher = "Korean Society for Molecular and Cellular Biology",
number = "4",

}

TY - JOUR

T1 - Fetal mouse selenophosphate synthetase 2 (SPS2)

T2 - biological activities of mutant forms in Escherichia coli.

AU - Kim, T. S.

AU - Yu, M. H.

AU - Chung, Y. W.

AU - Kim, Jae-Hong

AU - Choi, Eui Ju

AU - Ahn, K.

AU - Kim, Ick Young

PY - 1999/8/31

Y1 - 1999/8/31

N2 - A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.

AB - A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.

UR - http://www.scopus.com/inward/record.url?scp=0033620963&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033620963&partnerID=8YFLogxK

M3 - Article

VL - 9

SP - 422

EP - 428

JO - Molecules and Cells

JF - Molecules and Cells

SN - 1016-8478

IS - 4

ER -