Diaphorase and

The third study has employed 4,6-dinitrobenzofuroxan and as metabolic systems the one-electron reductants NADPHxytochrome P450 reductase and ferredoxin NADP(+) reductase and the two-electron reductants DT-diaphorase and Enterobacter cloacae nitroreductase [239]. The compound is activated either by DT-diaphorase or nitroreductase. 

Riley, R.J. and Workman, P., 1992, DT-diaphorase and cancer chemotherapy. Biochem. Pharmacol. 43 1657-1669... 

Testicular effects were also investigated after oral administration of 2000 mg/kg bw di(2-ethylhexyl) phthalate for seven consecutive days to 13-week-old male Wistar rats (Saxena et al., 1985). Degeneration was observed in about 40% of the seminiferous tubules. Loss of succinic dehydrogenase, NADH-diaphorase and acid phosphatase activity and increases in adenosine triphosphatase, glucose-6-phosphate dehydrogenase and alkaline phosphatase activity were observed in treated rats. 

Schmidt, H. H., Smith, R. M., Nakane, M., and Murad, F. (1992). Ca /calmodulin-dependent NO synthase type I A biopteroflavoprotein with Ca /calmodulin-inde-pendent diaphorase and reductase activities. Biochemistry 31, 3243-3249. 

Serum or bile samples (0.2 ml) are incubated with 100 mg arylamine glass bound to -hydroxysteroid dehydrogenase, 200 mg arylamine-glass-bound diaphorase and 0.5 ml colour reagent in a 15 ml conical flask, in the dark at 30°C for 15 min (standard assay conditions for mixture of immobilised enzyme) under continuous stirring. The A54o of the reaction mixture is read. 

DOM treatment also rapidly decreases cellular GSH, which precedes neurotoxicity. This decrease is primarily due to DOM-mediated GSH efflux. DOM also induces an increase in oxidative stress as indicated by increases in ROS and lipid peroxidation products, which follow GSH efflux. Astrocytes from both genotypes are resistant to DOM-mediated neurotoxicity and present a diminished Ca2+ response to DOM-mediated toxicity (Walser et al., 2006). Exposure of neonatal rat microglia to DOM triggers the release of TNF-a and matrix metalloproteinase-9 (MMP-9) (Mayer et al., 2001). These molecules are involved in the modulation of neuroinflammation in brain (Farooqui et al., 2007). Collective evidence suggests that DOM-mediated neurodegeneration involves changes in cellular redox, oxidative stress, and increased expression of cytokines, nitric oxide synthase, NADPH diaphorase, and matrix metalloproteinase-9 (Walser et al., 2006 Chandrasekaran et al., 2004 Ananth et al., 2003a,b Mayer et al., 2001). 

Ananth C., Dheen S. T., Gopalakrishnakone R, and Kaur C. (2003a). Distribution of NADPH-diaphorase and expression of nNOS, N-methyl-D-aspartate receptor (NMDARl) and non-NMDA glutamate receptor (GlutR2) genes in the neurons of the hippocampus after domoic acid-induced lesions in adult rats. Hippocampus 13 260-272. 

Marin A, Lopez de Cerain A, Hamilton E, Lewis AD, Martinez-Penuela JM, Idoate MA, Bello J. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours. Br J Cancer 1997 76 923-929. 

Since mainly the E° of the mediator dictates at what potential the heterogenous electron transfer occurs, the oxidation of NADH can now take place at a much lower potential. The different mediator structures used to produce CMEs for NADH oxidation at a decreased overpotential are summarized in Table I. As is seen in the table, not only chemically modified electrodes based on only immobilized redox mediators have been used for this purpose, but also electrodes based on the combination of redox mediators and NADH oxidizing enzymes (diaphorase and NADH dehydrogenase) as well as electrodes made of the conducting radical salts of tetrathiafulvalinium-7,7,8,8-tetracyanoquinodimethan (TTF-TCNQ) and W-methyl-phenazin-5-ium-7,7,8,8-tetracyanoquinodimethan (NMP-TCNQ). 

Figure 28.4. Mechanism of quinone drug redox cycling by liver microsomal NADPH cytochrome P450 reductase. A one-electron pathway competes with a two-electron process (catalyzed by DT-diaphorase) and is coupled with the monoxygenation and glucuronidation and sulfation systems.

Kinetic studies were carried out under anaerobic conditions in 0.05 M Tris-HCl buffer (pH 7.4) with Thunberg cuvettes and a 2mM stock solution of selected experimental agents dissolved in DMSO. The experimental agent was added to the top port and DT-diaphorase and NADH in the Tris buffer were added to the bottom. Thereafter both ports were purged with argon for 20 minutes and then equilibrated to 30°C. Ports were then mixed and the reaction monitored 25 minutes at... 

Boger m) showed that the transhydrogenase activity catalyzed by ferredoxin-NADP reductase obtained from Bumilleriopsis filiformis, which is very similar to the spinach enzyme, is regulated by ferredoxin and that one common nicotinamide nucleotide binding site is involved in both the diaphorase and the transhydrogenase reactions. 

Distribution of NADPH-diaphorase and expression of nNOS, N-methyl-D-aspartate receptor (NMDARl) and... 

GC mode experiments have been applied to the study of immobilized oxi-doreductases such as diaphorase and the glucose oxidase/glucose system (15,21), which has also been investigated by feedback with ferrocenyl mediators (10). In the feedback experiment, the catalytic cycle of the enzyme is dependent on ferrocenium species generated at the tip, as described in Sec. I.C. A generation-collection experiment utilizes a bulk solution concentration of the oxidized mediator, and the tip is poised at a potential sufficiently positive to detect ferrocene, or another reaction product such as H202, present near the interface (Fig. 6). The enzymatic reaction therefore occurs over the whole specimen, wherever there is active enzyme and a supply of substrate. In the feedback experiment, the tip current includes the flux of mediator from bulk solution as well as the flux of reduced mediator due to... 

Enzyme product signals can be amplified by enzymatic cycling systems. For example, as shown in Fig. 2, when alcohol dehydrogenase/diaphorase and... 

Sarlauskas J et al., Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds Their enzymatic reactions and cytotoxicity, Arch. Biochem. Biophys., 346, 219, 1997. 

Segura-Aguilar J, Lind C. 1989. On the mechanism of the Mnj -induced neurotoxicity of dopamine Prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase. Chem Biol Interact 72 309-324. 

Artificial donors include the viologen dyes and hydrosulfite. Methyl viologen can replace clostridial Fd or Fid. Oxidation of the monomer of poly-)S-hydroxybutyrate, a major component of soybean nodule bac-teroids, has been coupled to N2ase to give very low activity via a complex system composed of a dehydrogenase, NAD a diaphorase, and a viologen dye 14). Hydrosulfite is the most useful donor for in vitro N2ase assays since it can be used in substrate amounts in contrast to all other natural and artificial donors (22). 

The hexokinase reaction scheme used in the Bayer GLUCOMETER ENCORE test strip is shown later. Hexokinase, ATP, and magnesium react with glucose to produce glucose-6-phosphate. The glu-cose-6-phosphate reacts with glucose-6-phosphate dehydrogenase and NAD to produce NADH. The NADH then reacts with diaphorase and reduces the tetrazolium indicator to produce a brown compound (formazan). The reaction sequence requires three enzymes but is insensitive to oxygen. 

FIGURE 1. Restoration of NADPH-diaphorase and NADP photoreducing activities on reconstituted thylakoids. 

Polypyrrole diaphragm electrode coated with diaphorase and Co(phen)3 +... 

Oligosaccharide acceptor oxidase (oligosaccharide dehydrogenase), cytochrome hi, diaphorase, and fructose dehydrogenase can he employed in place of the above mentioned glucose dehydrogenase and laccase has heen replaced hy other phenol oxidizing enzymes [30],... 

Diaphorase and Cytochrome Reductase. Enzymes have been isolated from animal sources that have many of the properties of the various yeast enzymes. The first, and simplest, was liberated from particulate structures by Straub, who employed dilute ethanol and ammonium sulfate at 43 C. The enzyme could then be purified and was named a diaphorase. Diaphorase was coined to identify a widespread group of enzymes that transfer electrons from DPNH to dyes. Many of the purified flavoproteins have been found to oxidize pyridine nucleotides, and almost all of the flavoproteins can reduce dyes. Straub s diaphorase reduces methylene blue but not cytochrome c. Slight modification of the isolation procedure was found by workers at the Enzyme Institute of the University of Wisconsin to yield a cytochrome reductase. The relation between these preparations is not known, but it is possible that one is derived from the other. Cytochrome reductase contains 4 atoms of iron for each flavin, whereas Straub s preparation contains little iron. Both proteins have molecular weights around 75,000, and contain 1 equivalent of FAD. 

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