NADH diaphorase

Histochemical studies have demonstrated an alteration in NADPH- and NADH-diaphorase activity in the lungs of rats exposed to 0.8-ppm ozone for 7 days. ... 

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. 

In this test, p-hydroxybutyrate in the presence of NAD is converted by P-hydroxybutyrate dehydrogenase to acetoacetate, producing NADH. Diaphorase catalyzes the reduction of nitroblue tetrazolium (NBT) by NADH to produce a purple compound and its absorbance is read in a special meter that provides a digital readout. 

NADH diaphorase activity has been shown to be strong in human pinealocytes (BayerovA and Bayer 1967) and strong to moderate in rat pinealocytes in vivo (Bostelmann 1963, 1968, Tapp et al. 1973) and in vitro (TascA et al. 1968, S.Milcu et al. 1968), in rabbit pinealocytes (Bostelmann 1963) and in guinea pig pinealocytes (Vollrath and Schmidt 1969). 

At first, the enzymes involved were found to be flavoproteins, capable of transferring an electron from NADH or NADPH to an electron acceptor. NADH and NADPH cytochrome c reductase, as well as NADH diaphorase, were found to have transhydro-genase properties. The flavin nucleotide is not involved in the reaction, because it can be extracted from the protein without interfering with the transhydrogenation. Later, true transhydrogenases were found. A transhydrogenase has been purified from the ammonium sulfate fraction of rat liver mitochondria. Kaplan and his associates purified a transhydrogenase from heart mitochondria. 

An NADH diaphorase has been purified from human erythrocytes. The enzyme activity can be determined in a system containing 2.6 dichlorophenol-indo-phenol, Tris HCl buffer (pH 7.55), EDTA, and the enzyme. The reduction of 2.6 dichlorophenol-indo-phenol is followed spectrophotometrically. The purified enzyme contains one mole of a flavin adenine nucleotide (probably FAD) per 195,000 g of protein. The enzyme functions with either NADPH or NADH as hydrogen donor, but the affinity for NADH is almost 10 times greater than that for NADPH. 

Figure 8.9 Regeneration of NADH using diaphoras and electric power. Dl diaphorase [7a].

Ferricyanide has been employed as an electron acceptor for lactate in place of NAD Alternatively, diaphorase can be used in conjunction with ferricyanide Bindschedler s Green ferricenium or dichlorophenylindophenol to catalyze the oxidation of NADH while providing a more easily monitored species than NADH itself. 

While this anode is not useful in the context of implantable fuel cells, it is of interest because methanol is an attractive anodic fuel due to its availability and ease of transport and storage. The oxidation of one equivalent of methanol requires the reduction of three equivalents of NAD+ to NADH. As the NADH cofactor itself is not a useful redox mediator, a benzylviologen/diaphorase redox cycle, with a redox potential of 0.55 V vs SCE at pH 7, was used to regenerate NAD+ for use by the dehydrogenases, as depicted in Fig. 12.10. 

A colorimetric version of the method uses a tetrazolium salt. The oxidation of the NADH is coupled to the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide by a diaphorase (EC 1.6.4.3) and a deep blue formazan develops. The absorbance is read at a wavelength between 540 and 600 nm. This is more sensitive than the ultraviolet method but is more prone to interference, especially from any reducing agents present in the sample, which will result in a positive error. 

An alternative method uses glycerol dehydrogenase (EC 1.1.1.6) to produce NADH. This is measured either by its absorbance at 340 nm or by using a diaphorase (EC 1.6.4.3), which catalyses the reduction of a dye, p-iodonitro-tetrazolium violet (INT) to produce a coloured complex with an absorption maximum at 540 nm ... 

Eight human brain tumors contained DT-diaphorase, NADH cytochrome b5 reductase and NADPH cytochrome P450 reductase as assessed by enzyme activity and WB (Rampling et al., 1994). 

Palmore et al. applied the biocatalytic approach, utilizing the enzyme diaphorase (EC 1.6.4.3) to catalyze the reoxidation of NADH homogeneously, transferring electrons to a mediator, benzyl violo-gen. The mediator was then reoxidized at an electrode surface, with the overall scheme... 

Figure 3.13 — Set-up for the electrocatalytic oxidation of NADH catalysed by the reduced form of diaphorase (Dp). Dp (red) and Dp (ox) reduced and oxidized form of diaphorase. (Reproduced from [88] with permission of the Royal Society of Chemistry).

A nonmitochondrial muscle diaphorase has also been described (Koizumi and Brown (1971), capable of forming NOMb in solutions containing nitrite and Mb, using methylene blue as a nonspecific electron carrier. NOMb formation by both the mitochondrial and mtnmitochondrial diaphorase enzyme systems requires the presence of reduced NADH. 

The same procedure described for -hydroxysteroid dehydrogenase assay can be applied with a few differences [50]. Diaphorase (33 U/ml) is dissolved in 2 ml sodium carbonate-bicarbonate buffer (0.1 M, pH 9.0). Colour reagent is deprived of diaphorase NAD+ is replaced by NADH. The reaction is started by adding 0.1 ml diaphorase. 

DT diaphorase Dicoumarol-sensitive NADH/NADPH dye reductase, ductule small duct, dyspnea labored breathing. 

Fig. 11. Dissociation and reassociation of soluble, NAD-linked hydrogenase from Nocardia opaca. Diaphorase refers to NADH acceptor reductase activity. Reproduced, with permission, from Ref. 82.

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