Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins

Kei Miyano, Hiroki Kitahara, Shinobu Ohmi, Katsuko Kakinuma, Minoru Tamura

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Activation of the phagocyte NADPH oxidase involves assembly of p47 phox, p67 phox, Rac, and flavocytochrome b 558, and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1 min to 4 h at 25°C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b 558 became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t 1/2 = 6.6 h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.

Original languageEnglish
Pages (from-to)129-137
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume431
Issue number1
DOIs
Publication statusPublished - Nov 1 2004
Externally publishedYes

Fingerprint

NADPH Oxidase
Dilution
Oxidoreductases
Neutrophils
Fusion reactions
Proteins
Cell-Free System
Ethyldimethylaminopropyl Carbodiimide
Chemical activation
Amphiphiles
Flavin-Adenine Dinucleotide
Phagocytes
Half-Life
4-ethoxymethylene-2-phenyl-2-oxazoline-5-one
Western Blotting

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins. / Miyano, Kei; Kitahara, Hiroki; Ohmi, Shinobu; Kakinuma, Katsuko; Tamura, Minoru.

In: Archives of Biochemistry and Biophysics, Vol. 431, No. 1, 01.11.2004, p. 129-137.

Research output: Contribution to journalArticle

Miyano, Kei ; Kitahara, Hiroki ; Ohmi, Shinobu ; Kakinuma, Katsuko ; Tamura, Minoru. / Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins. In: Archives of Biochemistry and Biophysics. 2004 ; Vol. 431, No. 1. pp. 129-137.
@article{4c64033010944c25848eb34c6f404fbd,
title = "Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins",
abstract = "Activation of the phagocyte NADPH oxidase involves assembly of p47 phox, p67 phox, Rac, and flavocytochrome b 558, and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1 min to 4 h at 25°C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b 558 became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t 1/2 = 6.6 h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.",
author = "Kei Miyano and Hiroki Kitahara and Shinobu Ohmi and Katsuko Kakinuma and Minoru Tamura",
year = "2004",
month = "11",
day = "1",
doi = "10.1016/j.abb.2004.08.004",
language = "English",
volume = "431",
pages = "129--137",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins

AU - Miyano, Kei

AU - Kitahara, Hiroki

AU - Ohmi, Shinobu

AU - Kakinuma, Katsuko

AU - Tamura, Minoru

PY - 2004/11/1

Y1 - 2004/11/1

N2 - Activation of the phagocyte NADPH oxidase involves assembly of p47 phox, p67 phox, Rac, and flavocytochrome b 558, and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1 min to 4 h at 25°C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b 558 became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t 1/2 = 6.6 h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.

AB - Activation of the phagocyte NADPH oxidase involves assembly of p47 phox, p67 phox, Rac, and flavocytochrome b 558, and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1 min to 4 h at 25°C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b 558 became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t 1/2 = 6.6 h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.

UR - http://www.scopus.com/inward/record.url?scp=5344232045&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=5344232045&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2004.08.004

DO - 10.1016/j.abb.2004.08.004

M3 - Article

VL - 431

SP - 129

EP - 137

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -