Characterization of Mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis

Eun Joo Noh, Sang Wook Kang, Yong Jae Shin, Dong Chung Kim, In Sun Park, Min Young Kim, Boe Gwun Chun, Bon Hong Min

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma argininc dciminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370 ± 50 μM. Its optimal temperature and pH were 41°C and pH 6.4, respectively, and enzyme activity remained ≥ 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the cndotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

Original languageEnglish
Pages (from-to)137-143
Number of pages7
JournalMolecules and Cells
Volume13
Issue number1
Publication statusPublished - 2002 Feb 1

Fingerprint

Mycoplasma
Arginine
Nitric Oxide
Escherichia coli
Lipopolysaccharides
Terminator Codon
Native Polyacrylamide Gel Electrophoresis
Citrulline
Temperature
Sertoli Cells
Cytostatic Agents
DNA-Directed DNA Polymerase
Enzymes
Conditioned Culture Medium
Nitrites
Site-Directed Mutagenesis
Intraperitoneal Injections
Affinity Chromatography
Ammonia
Endotoxins

Keywords

  • Arginine
  • Arginine Deiminase
  • Endotoxemia
  • Lipopolysaccharide
  • Nitric Oxide

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Characterization of Mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis. / Noh, Eun Joo; Kang, Sang Wook; Shin, Yong Jae; Kim, Dong Chung; Park, In Sun; Kim, Min Young; Chun, Boe Gwun; Min, Bon Hong.

In: Molecules and Cells, Vol. 13, No. 1, 01.02.2002, p. 137-143.

Research output: Contribution to journalArticle

Noh, EJ, Kang, SW, Shin, YJ, Kim, DC, Park, IS, Kim, MY, Chun, BG & Min, BH 2002, 'Characterization of Mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis', Molecules and Cells, vol. 13, no. 1, pp. 137-143.
Noh, Eun Joo ; Kang, Sang Wook ; Shin, Yong Jae ; Kim, Dong Chung ; Park, In Sun ; Kim, Min Young ; Chun, Boe Gwun ; Min, Bon Hong. / Characterization of Mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis. In: Molecules and Cells. 2002 ; Vol. 13, No. 1. pp. 137-143.
@article{7508e3207f794289bd79154603004fa0,
title = "Characterization of Mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis",
abstract = "We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma argininc dciminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370 ± 50 μM. Its optimal temperature and pH were 41°C and pH 6.4, respectively, and enzyme activity remained ≥ 50{\%} for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the cndotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.",
keywords = "Arginine, Arginine Deiminase, Endotoxemia, Lipopolysaccharide, Nitric Oxide",
author = "Noh, {Eun Joo} and Kang, {Sang Wook} and Shin, {Yong Jae} and Kim, {Dong Chung} and Park, {In Sun} and Kim, {Min Young} and Chun, {Boe Gwun} and Min, {Bon Hong}",
year = "2002",
month = "2",
day = "1",
language = "English",
volume = "13",
pages = "137--143",
journal = "Molecules and Cells",
issn = "1016-8478",
publisher = "Korean Society for Molecular and Cellular Biology",
number = "1",

}

TY - JOUR

T1 - Characterization of Mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis

AU - Noh, Eun Joo

AU - Kang, Sang Wook

AU - Shin, Yong Jae

AU - Kim, Dong Chung

AU - Park, In Sun

AU - Kim, Min Young

AU - Chun, Boe Gwun

AU - Min, Bon Hong

PY - 2002/2/1

Y1 - 2002/2/1

N2 - We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma argininc dciminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370 ± 50 μM. Its optimal temperature and pH were 41°C and pH 6.4, respectively, and enzyme activity remained ≥ 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the cndotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

AB - We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma argininc dciminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370 ± 50 μM. Its optimal temperature and pH were 41°C and pH 6.4, respectively, and enzyme activity remained ≥ 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the cndotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

KW - Arginine

KW - Arginine Deiminase

KW - Endotoxemia

KW - Lipopolysaccharide

KW - Nitric Oxide

UR - http://www.scopus.com/inward/record.url?scp=0037187008&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037187008&partnerID=8YFLogxK

M3 - Article

C2 - 11911465

AN - SCOPUS:0037187008

VL - 13

SP - 137

EP - 143

JO - Molecules and Cells

JF - Molecules and Cells

SN - 1016-8478

IS - 1

ER -