NADPH oxidase/cytochrome

Superoxide is formed (reaction 1) in the red blood cell by the auto-oxidation of hemoglobin to methemo-globin (approximately 3% of hemoglobin in human red blood cells has been calculated to auto-oxidize per day) in other tissues, it is formed by the action of enzymes such as cytochrome P450 reductase and xanthine oxidase. When stimulated by contact with bacteria, neutrophils exhibit a respiratory burst (see below) and produce superoxide in a reaction catalyzed by NADPH oxidase (reaction 2). Superoxide spontaneously dismu-tates to form H2O2 and O2 however, the rate of this same reaction is speeded up tremendously by the action of the enzyme superoxide dismutase (reaction 3). Hydrogen peroxide is subject to a number of fates. The enzyme catalase, present in many types of cells, converts... 

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... 

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. 

LOX-dependent superoxide production was also registered under ex vivo conditions [55]. It has been shown that the intravenous administration of lipopolysaccharide to rats stimulated superoxide production by alveolar and peritoneal macrophages. O Donnell and Azzi [56] proposed that a relatively high rate of superoxide production by cultured human fibroblasts in the presence of NADH was relevant to 15-LOX-catalyzed oxidation of unsaturated acids and was independent of NADPH oxidase, prostaglandin H synthase, xanthine oxidase, and cytochrome P-450 activation or mitochondrial respiration. LOX might also be involved in the superoxide production by epidermal growth factor-stimulated pheochromo-cytoma cells [57]. 

O Donnell et al. [70] found that LOX and not cyclooxygenase, cytochrome P-450, NO synthase, NADPH oxidase, xanthine oxidase, ribonucleotide reductase, or mitochondrial respiratory chain is responsible for TNF-a-mediated apoptosis of murine fibrosarcoma cells. 15-LOX activity was found to increase sharply in heart, lung, and vascular tissues of rabbits by hypercholesterolemia [71], Schnurr et al. [72] demonstrated that there is an inverse regulation of 12/15-LOXs and phospholipid hydroperoxide glutathione peroxidases in cells, which balanced the intracellular concentration of oxidized lipids. 

Hexachloroethane is metabolized by the mixed function oxidase system by way of a two-step reduction reaction involving cytochrome P-450 and either reduced nicotinamide adenine dinucleotide phosphate (NADPH) or cytochrome b5 as an electron donor. The first step of the reduction reaction results in the formation of the pentachloroethyl free radical. In the second step, tetrachloroethene is formed as the primary metabolite. Two chloride ions are released. Pentachloroethane is a minor metabolic product that is generated from the pentachloroethyl free radical. 

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 ... 

The cytochrome (by virtue of its ability to accept and donate electrons during its function in electron transport) can exist in either the oxidised or the reduced state. In reduced-minus-oxidised difference spectra, it has absorption maxima at 426, 530 and 558 nm, typical of many b-type cytochromes. The ease with which the cytochrome can accept and donate electrons is expressed by its redox (reduction-oxidation) potential, which is measured in millivolts. Unlike most mammalian b cytochromes, which have much higher midpoint potentials, that of the cytochrome of the NADPH oxidase is -245 mV. Be-... 

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

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. 

The cytochrome b and flavin content and properties of the 02 -forming NADPH oxidase solubilized from activated neutrophils. Biochim. Biophys. Acta 746, 40-7. 

NADPH oxidase and cytochrome b559 in human neutrophils. Eur. J. Haematol. 43, 67-77. 

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. 

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. 

These observations were taken further by examining whether y-interferon treatment could up-regulate NADPH oxidase function in CGD neutrophils and monocytes. It was found that 12 out of 13 patients with autosomal recessive CGD had increased oxidase activity upon y-interferon exposure the only patient not responding was the one devoid of the b cytochrome. In X-linked CGD, 9 of 13 showed no improvement, whereas 3 showed some improvement and 1 had oxidase activity increased to near-normal levels. Patients with atypical X-linked CGD (i.e. low oxidase activity and some cytochrome b) appear to respond best to y-interferon treatment. Interferons-a and -ft are without affect. This enhancement of oxidase function (detected by NBT slide tests and O2 production) is due, at least in part, to increased levels of mRNA for the heavy chain of cytochrome b. In the absence of y-interferon treatment, monocyte-derived macrophages have extremely low or undetectable levels of mRNA for the cytochrome b heavy chain however, this is increased about fivefold (to about 5% of normal) after y-interferon treatment. 

Disulfoton induced the liver MFO system in animals (Stevens et al. 1973). In the same study, exposure to disulfoton orally for 3 days also increased ethylmorphine N-demethylase and NADPH oxidase activities, but had no effect on NADPH cytochrome c reductase. Thus, the induction of the MFO system required repeated dosing with relatively high doses. Furthermore, these changes are not specific for disulfoton exposure, and these subtle liver effects require invasive techniques in humans to obtain liver tissue for performance of these enzyme assays. 

Figure 17.26 Generation of the superoxide radical by NADPH oxidase. This Limited electron transfer chain is described in Chapter 9, where roles of FAD and cytochrome b are discussed. Red - reduced Ox - oxidised.

In addition to aconitases, nitric oxide is an inhibitor of many other enzymes such as ribonucleotide reductase [71], glutathione peroxidase [72,73], cytochrome c oxidase [74], NADPH oxidase [75], xanthine oxidase [76], and lipoxygenase [77] but not prostaglandin synthase [78]. (Mechanism of lipoxygenase inhibition by nitric oxide is considered in Chapter... 

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