Xanthine oxidase, cytochrome

In the xanthine/xanthine oxidase-cytochrome c method originally developed by McCord and Fridovich (M19), a typical assay mixture consists of oxidized1 cytochrome c, xanthine, sufficient xanthine oxidase, and phosphate buffer at pH 7.8 containing EDTA in a total volume of 3 ml. The rate of reaction is followed at 550 nm. One unit of SOD activity is defined as the amount that causes 50% inhibition of the rate of reduction of cytochrome c. 

In a study on the antioxidant effects of the components from H. diffusa on xanthine-xanthine oxidase cytochrome c, Lu et al. found that quercetin 3-0-sambubioside (22) and quercetin 3-0-sophoroside (23) are superoxide radical scavengers. The kaempferol glycoside (18) and the itidoid glycosides having cinnamoyl or feruloyl substituents on the glucose units [(31) and (33)], as well as asperuloside (48), were found to be inactive in this assay [29]. 

Several quinones are used clinically in the chemotherapy of cancer [61,62]. Some examples are adriamycin (doxorubicin), daunomycin (daunorubicin), mitomycin C and more recent diaziridinyl benzoquinones and diamino anthraquinones [5]. Physiological enzyme based reduction of these quinones caused by xanthine oxidase, cytochrome P450 reductase etc.[63], leads to the formation of semiquinone and hydroquinone forms. Pulse radiolysis can generate and characterise these intermediates and products [10]. 

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... 

McCord, J.M. and Fridovich, I. (1968) The reduction of cytochrome C by milk xanthine oxidase. J. Biol. Chem. 243, 5753-5760. 

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

K.V. Gobi, Y. Sato, and F. Mizutani, Mediatorless superoxide dismutase sensors using cytochrome c-modified electrodes xanthine oxidase incorporated polyion complex membrane for enhanced activity and in-vivo analysis. Electroanalysis 13, 397-403 (2001). 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

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. 

Superoxide-dismuting activity of copper rutin complex was confirmed by comparison of the inhibitory effects of this complex and rutin on superoxide production by xanthine oxidase and microsomes (measured via cytochrome c reduction and by lucigenin-amplified CL, respectively) with their effects on microsomal lipid peroxidation [166]. An excellent correlation between the inhibitory effects of both compounds on superoxide production and the formation of TBAR products was found, but at the same time the effect of copper rutin complex was five to nine times higher due to its additional superoxide dismuting capacity. 

Values (p.mol I-1) for Inhibitory Effects of Metal-Rutin Complexes and Rutin on Cytochrome c Reduction by Xanthine Oxidase (I), Iron-Catalyzed Microsomal Lipid Peroxidation (II), and Lucigenin-Amplified Microsomal CL (III) [167]... 

Possible errors due to the competition of cytochrome c reduction with the reversible reduction of quinones by superoxide are frequently neglected. For example, it has been found that quinones (Q), benzoquinone (BQ), and menadione (MD) enhanced the SOD-inhibitable cytochrome c reduction by xanthine oxidase [6]. This seems to be a mystery because only menadione may enhance superoxide production by redox cycling ( °p)"]/ [MD] =-0.20 V against ,0[02 ]/[02] 0.16 V) via Reactions (3) and (4), whereas for... 

In addition to binding to cytochrome c oxidase, cyanide inhibits catalase, peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, and succinic dehydrogenase activities. These reactions may make contributions to the signs of cyanide toxicity (Ardelt et al. 1989 Rieders 1971). Signs of cyanide intoxication include an initial hyperpnea followed by dyspnea and then convulsions (Rieders 1971 Way 1984). These effects are due to initial stimulation of carotid and aortic bodies and effects on the central nervous system. Death is caused by respiratory collapse resulting from central nervous system toxicity. 

The basis of this assay was first used to measure the activity of superoxide dismutase (SOD) using a xanthine/xanthine oxidase 02"-generating system. O2 generated via this enzyme will reduce feni (oxidised)-cytochrome c, but SOD (which has a much higher affinity for O2" than cytochrome c) will prevent this reduction. Babior, Kipnes and Cumutte (1973) modified this technique to provide a specific assay to measure O2 production by activated neutrophils. Thus, 02" reduces cytochrome c (measured by an absorbance increase at 550 nm), but this reduction will be blocked by the addition of exogenous SOD (Fig. 5.10). 

Komiyama, T., Kikuchi, T., and Sugiura, Y., 1986, Interactions of anticancer quinone dmgs, aclacinomycin A, adriamycin, carbazilquinone, and mitomycin C, with NADPH-cytochrome P-450 reductase, xanthine oxidase and oxygen, J. Pharmacobiodyru 9 651-664. 

Wenk M, Todesco L, Krahenbuhl S. Effect of St John s wort on the activities of CYP1A2, CYP3A4, CYP2D6, N-acetyltransferase 2, and xanthine oxidase in healthy males and females. Br J Clin Pharmacol 2004 57(4) 495 99. Wang LS, Zhou G, Zhu B, et al. St John s wort induces both cytochrome P450 3A4-catalyzed sulfoxidation and 2C19-dependent hydroxylation of omeprazole. Clin Pharmacol Therapeut 2004 75(3) 191-197. 

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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