A comparison of selected physical properties of hepatic sorbitol dehydrogenases [L-iditol: NAD oxidoreductases] from four mammalian species.

1. The sorbitol dehydrogenases [L-iditol: NAD oxidoreductase] from livers of cow, man, rat and sheep each possess molecular weights of about 140,000. The beef, rat and sheep liver enzymes are composed of subunits of molecular weight 40,000. 2. The sorbitol dehydrogenases from livers of these four species each possess an isoelectric point of 7.3. 3. The four enzyme preparations show identical mobilities upon disc-gel electrophoresis and yield a single band of enzymic activity. 4. Sorbitol dehydrogenase activity is activated by the presence of ampholines or by increasing ionic strengths, with maximal activation at about 0.5 M salt concentration. These factors may cause the Km for NAD to be lowered.     

Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves

Summary The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-membrane-bound quinone reductase activity are fully reduced hydroquinones rather than semi-quinone radicals. This peculiar kinetic property is common with soluble NAD(P)H-QR, plasma-membrane-bound NAD(P)H:quinone reductase purified from onion roots, and animal DT-diaphorase. These and previous results demonstrate that soluble and plasma-membrane-bound NAD(P)H:quinone reductases are strictly related flavo-dehydrogenases which seem to replace DT-diaphorase in plant tissues. Following purification to homogeneity, the soluble NAD(P)H-QR from tobacco leaves was digested. Nine peptides were sequenced, accounting for about 50% of NAD(P)H-QR amino acid sequence. Although one peptide was found homologous to animal DT-diaphorase and another one to plant monodehydroascorbate reductase, native NAD(P)H-QR does not seem to be structurally similar to any known flavoprotein.Abbreviations MDAR monodehydroascorbate reductase - PM plasma membrane - NAD(P)H-QR NAD(P)H:quinone oxidoreductase - DPI diphenylene iodonium - DQ duroquinone - CoQ₂ coenzyme Q₂     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit

NAD+-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The Km, values for sorbitol and fructose were 96.4+/-8.60 and 4239+/-33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.     

NAD(P)H:flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K(N), of 394 microM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Polymorphism of sorbitol dehydrogenase and 6-phosphogluconate dehydrogenase in swine (Sus scrofa)

More than 300 kidneys from the local bacon factory were investigated and it was shown that the enzyme sorbitol dehydrogenase (EC 1.1.1.14) exhibits a genetically determined polymorphism. While homozygous individuals yield only a single enzyme band after starch gel electrophoresis, heterozygotes show multiple bands.
On the same material also 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and lactate dehydrogenase were investigated, the former showed also clear genetic variants while the latter appeared only in one form.
Gene frequencies for different breeds and types of pigs are given and the results discussed.     

Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (Km(app)) was approximately 0.3 nM and the apparent maximum velocity (Vmax(app)) was 16 U mg(-1). Substrate inhibition was observed above 5 nM. NADH was the electron donor, Km(app)= 340 microM and Vmax(app) = 46 Umg(-1). FAD was also a cofactor with a Km(app) of 3.1 microM and Vmax(app) of 89 U mg(-1). The turnover number for NADH oxidation was 25 s(-1). Possible physiological roles of the Fe(III) reduction by this enzyme are discussed.     

NADH/NAD+ interaction with NADH: ubiquinone oxidoreductase (complex I)

The quantitative data on the binding affinity of NADH, NAD(+), and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.     

NAD(P)H: quinone oxidoreductase 1 expression in kidney podocytes.

NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase; DTD) is a cytosolic two-electron reductase, and compounds of the quinone family such as mitomycin C are efficiently bioactivated by this enzyme. The observation that DT-diaphorase is highly expressed in many cancerous tissues compared to normal tissues has provided us with a potentially selective target that can be exploited in the design of novel anticancer agents. Because of the relative lack of information about the cell-specific expression of DT-diaphorase, the purpose of this study was to map the distribution of this enzyme in normal human tissues. Fifteen tissue samples from normal human kidney were analyzed for expression of DT-diaphorase by immunohistochemistry (two-step indirect method). We found a specific high expression of DT-diaphorase in glomerular visceral epithelial cells (podocytes). These results suggest that a high expression of DT-diaphorase in podocytes could play a major role in the pathogenesis of renal toxicity and mitomycin C-induced hemolytic uremic syndrome, in which injury to the glomerular filtration mechanism is the primary damage, leading to a cascade of deleterious events including microangiopathic hemolytic anemia and thrombocytopenia. This observation has potential therapeutic implications because the DT-diaphorase metabolic pathway is influenced by many agents, including drugs, diet, and environmental cell factors such as pH and oxygen tension.     

Role of epoxide hydrolase, NAD(P)H:quinone oxidoreductase, cytochrome P450 2E1 or alcohol dehydrogenase genotypes in susceptibility to colorectal cancer

Colorectal cancer (CRC) is one of the most common forms of cancer in Western countries. CRC has been associated with genetic and lifestyle factors. Individual susceptibility to CRC may be due partly to variations in detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes may result in variations in detoxification activities, which subsequently might influence the levels of toxic/carcinogenic compounds, and this may influence the risk for CRC. Therefore, we determined whether polymorphisms in the genes coding for microsomal epoxide hydrolase (EPHX1), NAD(P)H:quinone oxidoreductase (NQO1), cytochrome P450 2E1 (CYP2E1) and alcohol dehydrogenase (ADH3) predispose to the development of CRC. DNA samples were obtained from 371 patients with sporadic CRC and 415 healthy controls. Patients and controls were all of Caucasian origin. All genetic polymorphisms were determined by polymerase chain reaction, eventually followed by restriction-fragment-length-polymorphism analyses, except for the EPHX1 codon 113 polymorphism, which was genotyped by an allele-specific discrimination assay. Calculation of crude Odds Ratios (ORs) revealed an increased risk for CRC associated with variant NQO1 (OR 1.5, 95% CI 1.1-2.0) and CYP2E1 intron 6 genotypes (OR 2.2, 95% CI 1.3-3.8). However, after adjustment for age and gender, logistic regression analyses only showed a statistically significant risk for CRC associated with variant NQO1 genotypes (OR 1.6, 95% CI 1.03-2.4). No associations were found between CRC and the other polymorphic genes as mentioned above. In conclusion, these data suggest that the presence of variant NQO1 genotypes, with expected reduced enzyme activities might enhance susceptibility to CRC.     

Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues

Despite the extensive interest in NADPH:quinone oxidoreductase (NQO1, DT-diaphorase), there is little immunohistochemical information regarding its distribution in either normal human tissues or in human tumors. Using immunohistochemistry (IHC), we have examined cell-specific expression of NQO1 in many normal tissues and tumors as a step toward defining the distribution of NQO1 in humans. NQO1 was detected by IHC in respiratory, breast duct, thyroid follicle, and colonic epithelium, as well as in the corneal and lens epithelium of the eye. NQO1 was also detected by IHC in vascular endothelium in all tissues examined. NQO1 could also readily be detected in the endothelial lining of the aorta but was not detected using immunoblot analysis in the myocardium. Adipocytes stained positive for NQO1, and the enzyme was also detected by both IHC and immunoblot analysis in parasympathetic ganglia in the small intestine and in the optic nerve and nerve fibers. NQO1 was not highly expressed in five different human liver samples using immunoblot analysis, whereas studies using IHC demonstrated only trace NQO1 staining in isolated bile duct epithelium. NQO1 expresion was also examined by IHC in a variety of solid tumors. Marked NQO1 staining was detected in solid tumors from thyroid, adrenal, breast, ovarian, colon, and cornea and in non-small cell lung cancers. The NQO1 content of many solid tumors supports the use of NQO1-directed anticancer agents for therapeutic purposes, but the distribution of NQO1 in normal tissues suggests that potential adverse effects of such agents need to be carefully monitored in preclinical studies.