Article (Scientific journals)
Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity
Nguyen, Minh Vu Chong; Zhang, Leilei; Lhomme, Stanislas et al.
2012In Biochemical and Biophysical Research Communications, 419 (3), p. 453-458
Peer reviewed
 

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Keywords :
Cell-free protein expression; Diaphorase activity; NADPH oxidase Nox4; Truncated recombinant proteins; Cell-Free System; Cytosol; Escherichia coli; HEK293 Cells; Humans; NADH Dehydrogenase; NADPH Oxidase; Protein Structure, Tertiary; Recombinant Proteins
Abstract :
[en] The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7. nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8. nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6. nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2. nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain. © 2012 Elsevier Inc..
Disciplines :
Biochemistry, biophysics & molecular biology
Identifiers :
eid=2-s2.0-84862790990
Author, co-author :
Nguyen, Minh Vu Chong ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Zhang, Leilei;  GREPI AGIM FRE 3405, CNRS-UJF, Université Joseph Fourier, Grenoble, France
Lhomme, Stanislas;  PX'Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble, France
Mouz, Nicolas;  PX'Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble, France
Lenormand, Jean-Luc;  HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Université Joseph Fourier, UFR de Médecine, Domaine de la Merci, 38706 La Tronche, France
Lardy, Bernard;  GREPI AGIM FRE 3405, CNRS-UJF, Université Joseph Fourier, Grenoble, France
Morel, Françoise;  GREPI AGIM FRE 3405, CNRS-UJF, Université Joseph Fourier, Grenoble, France
External co-authors :
yes
Language :
English
Title :
Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity
Publication date :
2012
Journal title :
Biochemical and Biophysical Research Communications
ISSN :
0006-291X
Volume :
419
Issue :
3
Pages :
453-458
Peer reviewed :
Peer reviewed
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