NADPH oxidase components

The 02 -generating NADPH oxidase comprises five fundamental components  

Neutrophil priming Regulation during inflammatory activation 

Messenger RNA molecules for both subunits of the cytochrome and the two cytosolic components are detectable in unstimulated bloodstream cells. Experiments involving incubation of neutrophil suspensions with the protein synthesis inhibitor cycloheximide indicate that constitutive expression of one or more components of the oxidase is required for the neutrophil to maintain its ability to generate reactive oxidants. For example, when neutrophils are incubated in vitro with cycloheximide, their ability to generate reactive oxidants declines more rapidly than in control cells, as they age in culture (Fig. 7.12). This decline in oxidase activity when protein biosynthesis is blocked is not due to cell death, because cells treated with cycloheximide for this time still exclude trypan blue. Furthermore, when protein biosynthesis is stimulated in neutrophils by the addition of GM-CSF for 24 h in vitro, the ability to generate reactive oxidants is enhanced considerably above the levels observed in untreated cells. 

Incubation with y-interferon (2-50 U/ml) increases the ability of neutrophils to generate reactive oxidants in response to fMet-Leu-Phe, con A and PM A. Incubation times in excess of 2-4 h are required, and this enhanced activity is prevented if the cells are co-incubated with cycloheximide. This 

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Heyworth, PC., Bohl, B.P, Bokoch, G.M. and Curnutte, J.T. (1994). Rac translocates independently of the neutrophil NADPH oxidase components p47P and p07pho Evidence for its interaction with flavocytochromehssg./. Biol. Chem. 269, 30749-30752. 

Waite, K. A., R. WaUin, D. QuaUiotine-Mann, and L. C. McPhail. 1997. Phosphatidic acid-mediated phosphorylation of the NADPH oxidase component p47-phox. Evidence that phosphatidic acid may activate a novel protein kinase. 272 (24) 15569-78. 

Wientjes, F. B., A. W. Segal, and J. H. Hartwig. 1997. Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane. J Leukoc Biol 61 (3) 303-12. 

Jones SA, O Dormell VB, Wood JD, Broughton JP, Hughes EJ, Jones OT. Expression of phagocyte NADPH oxidase components in human endothelial cells. Am J Physiol 1996 271 H1626-H1634. 

NADPH-oxidase j 2O2 + NADPH 20p + NADP -F H+ Key component of the respiratory burst Deficient in chronic granulomatous disease... 

The electron transport chain system responsible for the respiratory burst (named NADPH oxidase) is composed of several components. One is cytochrome 6558, located in the plasma membrane it is a heterodimer, containing two polypeptides of 91 kDa and... 

Mutations in the Genes for Components of the NADPH Oxidase System Cause Chronic Granulomatous Disease... 

Knoller, S., Shpungin, S., and Pick, E. (1991) The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559./. Biol. Chem. 266, 2795-2804. 

The NADPH oxidase is in fact a multicomponent enzyme system that constitutes an electron transport chain from NADPH to O2. The components of this oxidase complex are now almost completely defined, and experiments performed primarily with CGD neutrophils have helped to identify these major constituents. 

For many years, the literature was confused with various reports of purified NADPH oxidase preparations , which contained a variety of unidentified components. Here, again, the CGD neutrophil has proved to be invaluable defective activity or amount of a putative oxidase component in the CGD neutrophil is very strong evidence that the component is a genuine constituent... 

In the early 1960s in Japan, a b-type cytochrome was found in horse neutrophils and, because it bound CO, it was proposed to be functional during the respiratory burst. This work went largely unnoticed, but in 1978 Segal and Jones in the United Kingdom discovered that a b-type cytochrome became incorporated into phagolysosomes furthermore, this cytochrome was absent in some patients with CGD. These workers correctly proposed that it was a key component of the NADPH oxidase. This cytochrome was a landmark discovery in phagocyte research for a number of reasons ... 

At around the same time as these discoveries, several groups were trying to determine the ways in which the NADPH oxidase could be activated in vitro (i.e. activated in broken-cell suspensions). The ultimate aim of such studies was to determine the minimal components necessary for assembly and activation of the oxidase in in vivo experiments. Thus, once these minimal constituents were identified, oxidase activity could then be reconstituted in vitro from the individual component parts. The first breakthrough in these studies was... 

Abo, A., Boyhan, A., West, I., Thrasher, A. J., Segal, A. W. (1992). Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components p67-phox, p47-phox, p2lracl, and cytochrome b.245. J. Biol. Chem. 267, 16767-70. 

Volpp, B. D., Nauseef, W. M Donelson, J. E Moser, D. R Clark, R. A. (1989). Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human respiratory burst oxidase. Proc. Natl. Acad. Sci. USA 86, 7195-9. Watson, F., Robinson, J. J., Edwards, S. W. (1991). Protein kinase C dependent and independent activation of the NADPH oxidase of human neutrophils. J. Biol. Chem. 266, 7432-9. 

Cassatella, M. A., Bazzoni, F., Calzetti, F., Guasparri, I., Rossi, F., Trinchieri, G. (1991). Interferon-/transcriptionally modulates the expression of the genes for the high affinity IgG-Fc receptor and the 47-kDa cytosolic component of NADPH oxidase in human polymorphonuclear leukocytes. J. Biol. Chem. 266, 22079-82. 

Extensive studies into the association of this cytochrome b with CGD neutrophils were performed by Segal and Jones, and by other workers, in the late 1970s and early 1980s. The cytochrome was completely absent (as determined by the absence of a distinctive absorption spectrum in spectroscopic studies) in almost all cases of X-linked CGD, but present at decreased levels in female relatives of these patients. In almost all cases of autosomal recessive CGD, the cytochrome was present but non-functional, in that it did not become reduced upon cellular activation. This indicated both the heterogeneous nature of the disease and also that some other biochemical defect was responsible for impaired function in these patients. Hence, the search was on for other components of the NADPH oxidase. 

Figure 5.22 NADPH oxidase complex. gp22 and gp91 form a heterodimer p40, p47 and p67 are accessory proteins. Phosphorylation of the accessory proteins causes aggregation of the components and activation of the oxidase activity...

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