Assessment of the flavoprotein nature of the redox core of neutrophil NADPH oxidase.
The latent NADPH oxidase activity of purified cytochrome b(558) from rabbit peritoneal neutrophils was expressed in a cell-free system consisting of either gel-filtrated cytosol from resting neutrophils, or a mixture of the three cytosolic activation factors, namely p47, p67 and the G protein Rac1. The cell-free system was supplemented with arachidonic acid and GTPgammaS. With gel-filtrated cytosol, the oxidase activity was relatively high (22 moles O(2)(-)/s/mole heme b in the absence of added FAD), and enhanced by less than one fourth upon addition of FAD. In contrast, with the purified cytosolic activation factors the rate of O(2)(-) production was low (8 moles O(2)(-)/s/mole heme b), and enhanced more than two-fold by a saturating concentration of FAD. The specificity of FAD was demonstrated by the lack of effect of FMN. FAD was determined together with heme b and the oxidase activity in eluates from a Sepharcryl column at the last step of the purification of cytochrome b(558). In the eluted fraction that contained both the maximal inducible oxidase activity and the highest amount of heme b, the molar amount of FAD was 20 times less than that of heme b. It is concluded that cytochrome b(558) is an NADPH-dependent flavocytochrome oxido-reductase (NADPH oxidase) in which one part of FAD is firmly bound and another, loosely attached. On the other hand, there may exist a parallel pathway of electron transfer from NADPH via distinct FAD dehydrogenase(s) to the heme b component of the NADPH oxidase.
Escriou
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Laboratoire de Biochimie (URA 1130/CNRS), Département de Biologie Moléculaire et Structurale, Grenoble, France.
Laporte
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Vignais
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D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism / Q000494:pharmacology; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism*
D003573:Cytochrome b Group; D005420:Flavoproteins; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases