Reference : Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibi...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/10993/27268
Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity
English
Nguyen, Minh Vu Chong mailto [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]
2012
Biochemical and Biophysical Research Communications
419
3
453-458
Yes
International
0006291X
[en] 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
[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..
http://hdl.handle.net/10993/27268
10.1016/j.bbrc.2012.01.136

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