Diaphorases

Neuropeptide Y. Neuropeptide Y [82785 5-3] (NPY) (255) is a 36-amiao acid peptide that is a member of a peptide family including peptide YY (PYY) [81858-94-8, 106338-42-5] (256) and pancreatic polypeptide (PPY) [59763-91-6] (257). In the periphery, NPY is present in most sympathetic nerve fibers, particulady around blood vessels and also in noradrenergic perivascular and selected parasympathetic nerves (66). Neurons containing NPY-like immunoreactivity ate abundant in the central nervous system, particulady in limbic stmctures. Coexistence with somatostatin and NADPH-diaphorase, an enzyme associated with NO synthesis, is common in the cortex and striatum. 

The requirement for reduction prior to DNA alkylation and crosslinking was first demonstrated by Iyer and Szybalski in 1964 [29], and can be induced both by chemical reducing agents such as sodium dithionite and thiols in vitro and by various reductive enzymes such as DT-diaphorase (NAD(P)H-quinone oxidoreduc-tase) in vitro and in vivo [47]. Much work to characterize the mechanism of reductive activation and alkylation has been carried out, principally by the Tomasz and Kohn groups, and Figure 11.1 illustrates a generally accepted pathway for mitomycin C [16, 48-50] based on these experiments, which is very similar to the mechanism originally proposed by Iyer and Szybalski [29]. 

NADPH-diaphorase activity is the ability of an enzyme to reduce soluble tetrazolium salts to an insoluble, visible formazan. This activity is being used by many laboratories to localize NO synthase histochemically. 

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

The reaction may be followed by measuring the rate of appearance or disappearance of MADH at 340 nm. In some methods a coupled reaction using diaphorase or phenazine methosulfate plus an indicator dye are used. The amount of pyruvate formed can also be measured colorimetrically. With the general avail-... 

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. 

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. 

Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R. (1991). Neuronal NADPH diaphorase is a nitric oxide synthetase. Proc. Natl Acad. Sci. USA 88, 2811-2814. 

Alonso J., Arevalo R., Garciaojeda E., Porteros A., et al. (1995). NADPH-diaphorase active and calbindin d-28k-immunoreactive neurons and fibers in the olfactory-bulb of the hedgehog (Erinaceus europaeus). J Comp Neurol 351, 307-327. 

The continuing interest in bioreductive alkylation is largely due to the clinical success of mitomycin C and the low reduction potentials observed in many tumors.9 The low reduction potentials favor the quinone to hydroquinone conversion necessary for bioreductive alkylation. Hypoxia due to low blood flow3 and/or the unusually high expression of the quinone two-electron reducing enzyme DT-diaphorase in some histological cancer types10-14 contribute to the tumor s tendency to reduce quinones. 

Selective bioreductive alkylation in high DT-diaphorase cancer types (melanoma renal and nonsmall-cell lung cancers)15 would exhibit maximal antitumor activity with minimal side effects. 

Belcourt, M.F. Hodnick, W. F. Rockwell, S. Sartorelli, A. C. Bioactivation of mitomycin antibiotics by aerobic and hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase. Biochem. Pharm. 1996, 51, 1669-1678. 

Gutierrez, P. L. The role of NAD(P)H oxidoreductase (DT-diaphorase) in the bioactivation of quinone-containing antitumor agents a review. Free Radio. Biol. Med. 2000, 29, 263-275. 

Rauth, A. M. Goldberg, Z. Misra, V. DT-diaphorase possible roles in cancer chemotherapy and carcinogenesis. Oncol. Res. 1997, 9, 339-349. 

Workman, P. Enzyme-directed bioreductive drug development revisted a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase. Oncol. Res. 1994, 6, 461 175. 

Vincent, S. R., Satoh, K, Armstrong, D. M. Fibiger, H. C. (1983). NADPH-diaphorase a selective histochemical marker for the cholinergic neurons of the pontine reticular formation. Neurosci. Lett. 43, 31-6. 

Hilbig, H. Punkt, K. (1997). 24-hour rhythmicity of NADPH-diaphorase activity in the neuropil of rat visual cortex. Brain Res. Bull. 43, 337-40. 

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. 

L.L. Moroz, T.P. Norekian, T.J. Pirtle, K.J. Robertson, and R.A. Satterlie, Distribution of NADPH-diaphorase reactivity and effects of nitric oxide on feeding and locomotory circuitry in the pteropod mollusc, Clione limacina. J. Comp. Neurol. 427, 274-284 (2000). 

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. 

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