Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

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• 作者：Minh Vu Chuong Nguyen^a ; ^{mvchuong@yahoo.fr} ; Leilei Zhang^a ; Stanislas Lhomme^b ; Nicolas Mouz^b ; Jean-Luc Lenormand^c ; Bernard Lardy^a ; Franç ; oise Morel^a
• 关键词：NADPH oxidase Nox4 ; Cell-free protein expression ; Diaphorase activity ; Truncated recombinant proteins
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 期刊代码：159_0006291x
• 类别：bio
• 出版时间：16 March, 2012
• 卷：419
• 期：3
• 页码：453-458
• 文件大小：554 K

摘要

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 卤 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 卤 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 卤 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 卤 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.