NADPH oxidase cell-free activation system

The second major breakthrough in understanding the defect in CGD neutrophils came through the development of assays in which the NADPH oxidase can be activated in a cell-free system in vitro ( 5.3.2.3). In these systems, activation of the oxidase can be achieved by the addition of cytoplasm to plasma membranes in the presence of NADPH and arachidonic acid (or SDS or related substances). Interestingly, the oxidase cannot be activated in these cell-free systems using extracts from CGD neutrophils however, cytosol and plasma membranes from normal and CGD neutrophils may be mixed, and in most cases activity is restored if the correct mixing pattern is used. For example, as may be predicted, in X-linked CGD it is the membranes that are defective (because the cytochrome b is deficient), whereas in autosomal recessive CGD the cytosol is defective in the cell-free system. 

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. 

Heyneman, R. A., Vercauteren, R. E. (1984). Activation of a NADPH oxidase from horse polymorphonuclear leukocytes in a cell-free system. J. Leuk. Biol. 36, 751-9. 

Other roles of arachidonic acid include activation of the NADPH oxidase in the cell-free system (see 5.3.2.3). It may be required for membrane fusion during the process of recruitment of oxidase and receptor molecules from the membranes of specific granules. Finally, there is some evidence that arachidonic acid may ... 

Aharoni, I., Pick, E. (1990). Activation of the superoxide-generating NADPH oxidase of macrophages by sodium dodecyl sulfate in a soluble cell-free system Evidence for involvement of a G protein. J. Leuk. Biol. 48,107-15. 

The molecular mechanism of the NO pro-inflammatory activity is also multifaceted NO regulates the inflammatory responses by cell-specific inhibition of the transcription factor NF-kB, IL-ip, interferon-y (IFNy). At sites of inflammation, increased free radical activity is associated with the activation of the neutrophil NADPH-oxidase and/or the uncoupling of a variety of redox systems, leading to a substantial increase in ROS. Free radicals thus produced, have the capacity to mediate tissue destruction, either alone or in concert with proteases [53]. 

Fig. 2. Interplay among superoxide anion, nitric oxide, and eicosanoids in high oxidative stress. The biological function of nitric oxide in target cells is influenced by the cellular redox state. In increased oxidative stress, which results in an oxidizing environment, NO readily form free radicals, including the highly reactive peroxynitrite (OONO ). Peroxynitrite can influence eicosanoid synthesis by interfering with different enzyme systems of the arachidonic acid cascade. Increased free radicals may also catalyze nonenzymic peroxidation of membrane PUFA (e.g., arachidonic acid), resulting in increased production of isoprostanes that possess potent vasoconstrictor activity. PLA, phospholipase NO, nitric oxide NOS, nitric oxide synthase NADPH oxidase, vascular NAD(P)H oxidase 02 , superoxide anion PUFA, polyunsaturated fatty acids EPA, eicosapentaenoic acid DHA, docosahexaenoic acid COX, cyclooxygenase PGI2 synthase, prostacyclin synthase.

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