Menadione Reductase Activity Research Articles

Higher activity of polymorphic NAD(P)H:quinone oxidoreductase in liver cytosols from blacks compared to whites

In human liver, the two-electron reduction of quinone compounds, such as menadione is catalyzed by cytosolic carbonyl reductase (CBR) and NAD(P)H:quinone oxidoreductase (NQO1) activities. We assessed the relative contributions of CBR and NQO1 activities to the total menadione reducing capacity in liver cytosols from black ( n = 31) and white donors ( n = 63). Maximal menadione reductase activities did not differ between black (13.0 ± 5.0 nmol/min mg), and white donors (11.4 ± 6.6 nmol/min mg; p = 0.208). In addition, both groups presented similar levels of CBR activities (CBR blacks = 10.9 ± 4.1 nmol/min mg) versus CBR whites = 10.5 ± 5.8 nmol/min mg; p = 0.708). In contrast, blacks showed higher NQO1 activities (two-fold) than whites (NQO1 blacks = 2.1 ± 3.0 nmol/min mg versus NQO1 whites = 0.9 ± 1.6 nmol/min mg, p < 0.01). To further explore this disparity, we tested whether NQO1 activity was associated with the common NQO1 * 2 genetic polymorphism by using paired DNA samples for genotyping. Cytosolic NQO1 activities differed significantly by NQO1 genotype status in whites (NQO1 whites[ NQO1*1/*1] = 1.3 ± 1.7 nmol/min mg versus NQO1 whites[ NQO1*1/*2+NQO1*2/*2] = 0.5 ± 0.7 nmol/min mg, p < 0.01), but not in blacks (NQO1 blacks[ NQO1*1/*1] = 2.6 ± 3.4 nmol/min mg versus NQO1 blacks[ NQO1*1/*2] = 1.1 ± 1.2 nmol/min mg, p = 0.134). Our findings pinpoint the presence of significant interethnic differences in polymorphic hepatic NQO1 activity.

Menadione enhances oxyradical formation in earthworm extracts: vulnerability of earthworms to quinone toxicity

NAD(P)H-cytochrome c reductase activities have been determined in the earthworms, L. rubellus and A. chlorotica, extracts. Menadione (0.35 mM, maximum concentration tested) was found to stimulate the rates of NADPH- and NADH-dependent cytochrome c reduction by three- and twofold, respectively. Superoxide dismutase (SOD) inhibited completely this menadione-mediated stimulation, suggesting that O 2 − is involved in the redox cycling of menadione. However, SOD had no effect on the basal activity (activity in the absence of quinone) in the case of NADH-dependent cytochrome c reduction, whereas it partially inhibited the basal activity of NADPH-cytochrome c reduction. This indicates direct electron transfer in the former case and the formation of superoxide anion in the latter. DT-diaphorase, measured as the dicumarol-inhibitable part of menadione reductase activity, was not detectable in the earthworms' extracts. In contrast, it was found that DT-diaphorase represents about 70% of the menadione reductase activities in the freshwater mussel, Dreissena polymorpha. The results of this work suggest that earthworms, compared with mussels, could be more vulnerable to oxidative stress from quinones due to lack, or very low level of DT-diaphorase, an enzyme considered to play a significant role in the detoxification of quinones. On the contrary, mussels have efficient DT-diaphorase, which catalyzes two-electron reduction of menadione directly to hydroquinone, thus circumventing the formation of semiquinone.

Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing.

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2 by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione-catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione-catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coli. Menadione-catalyzed LC was sensitive to antibiotic action. A decrease in menadione-catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione-catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.

Effects of the agrochemicals butachlor, pretilachlor and isoprothiolane on rat liver xenobioticmetabolizing enzymes

1. The herbicides butachlor (2-chloro-2′,6′,diethyl-N-[buthoxymethyl] acetanilide) and pretilachlor (2-chloro-2′,6′-diethyl-N-[2-propoxyethyl] acetanilide) are widely used in Asia, South America, Europe and Africa. Isoprothiolane (diisopropyl-1,3-dithiolan-2-ylidenemalonate) is used as a fungicide and an insecticide in rice paddies. We administered these agrochemicals to the male rat and examined their effects on cytochrome P450 (P450), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT), and NAD(P)H-quinone oxidoreductase 1 (NQO1)-related metabolism in the liver. 2. Administration of isoprothiolane, butachlor or pretilachlor to rat induced hepatic P4502B subfamily-dependent enzyme activities (pentoxyresorufin O-depentylation and testosterone 16 β-hydroxylation) up to 271-413% of control, which coincided with the increase in expression levels of the P4502B apoprotein. 3. Activities of GST toward 1-chloro-2,4-nitrobenzene and 3,4-dichloronitrobenzene were slightly induced (127-133% of control) in the liver of the rat treated with these pesticides. On the other hand, marked elevations of UDPGT activities toward p-nitrophenol (164-281% of control) were observed. NQO1-related metabolism (menadione reductase activity) was also induced (123-176% of control) in the liver of rat treated with these agrochemicals. 4. These results indicate that some of the agrochemicals currently in use are capable of inducing phase I and II xenobiotic-metabolizing enzyme activities in an isozyme selective manner. The induction of these activities may disrupt normal physiologic functions related to these enzymes in exposed animals.

Carbonyl reductase purified from rabbit liver is not the product of a carbonyl reductase gene (RCBR5 or RCBR6) cloned from the rabbit liver cDNA library.

Six peptides were obtained by the digestion of carbonyl reductase purified from rabbit liver. The amino acid sequences of the six peptides were virtually identical to the corresponding regions in amino acid sequences deduced from two cloned carbonyl reductase genes (RCBR5 and RCBR6). However, there was a difference of one amino acid residue between the sequences of peptides from the purified enzyme and the corresponding region in the amino acid sequences deduced from the two cDNAs. The purified carbonyl reductase was confirmed to exhibit no reactivity towards menadione, even though the transient expression of the two cDNA for rabbit liver carbonyl reductase has been reported to cause a marked increase of menadione reductase activity in COS7 cells. The enzyme purified from rabbit liver was inactivated by thiol-specific reagents, 5,5'-dithiobis(2-nitrobenzoic acid) and sodium tetrathionate, suggesting that menadione probably interacts with the functional cysteine residue(s), and cannot serve as a substrate of the purified enzyme. Based on these results, it is concluded that the carbonyl reductase purified from rabbit liver is not the product of cloned carbonyl reductase gene (RCBR5 or RCBR6).

Heterologous expression of carbonyl reductase: Demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance

Transfection of murine NIH3T3 fibroblasts with a pSV2-derived eukaryotic expression vector for human cytosolic carbonyl reductase (E.C. 1.1.1.141) resulted in clones with increased carbonyl reductase activity as demonstrated by an elevation in cellular NADPH-dependent alcohol (menadione) reductase activity. Prostaglandin 9-ketoreductase (9KR) activity, previously noted only in purified enzyme preparations, was also elevated. Although the cellular molar capacity of 9KR activity was less than menadione reductase activity (picomoles versus nanomoles per mg of protein), when compared to endogenous activity there was a greater relative increase in 9KR activity as compared to menadione activity (10 fold increase versus 3 fold). Thus, the 9KR properties of carbonyl reductase may have a physiologic role in prostaglandin regulation. Most transgenic clones lost their enhanced carbonyl reductase activity despite continuous selection, but two clones retained enhanced enzyme activity. RNA analysis indicated that these two murine clones expressed human carbonyl reductase mRNA. These two clones overexpressing carbonyl reductase did not display resistance to menadione, in agreement with a previous report. There was, however, a demonstrable increase in resistance to paraquat of a magnitude similar to that previously noted with transgenic cell lines overexpressing manganese Superoxide dismutase.

Responses of hepatic biotransformation and antioxidant enzymes to CYP1A-inducers (3-methylcholanthrene, β-naphthoflavone) in sea bass ( Dicentrarchus labrax), dab ( Limanda limanda) and rainbow trout ( Oncorhynchus mykiss)

The time-course responses of hepatic biotransformation and antioxidant enzymes to cytochrome P4501A-inducers in fish were examined using marine and freshwater species, viz. respectively, sea bass ( Dicentrarchus labrax) and dab ( Limanda limanda) exposed up to 5 to 11 days to 3-methylcholanthrene (3MC; intraperitoneal (i.p.) 20 mg kg −1), and rainbow trout ( Oncorhynchus mykiss) exposed up to 15 days to β-naphthoflavone (BNF; i.p. 5 mg kg −1). The enzymes included those which are part of the mammalian [ Ah]-gene locus, viz. cytochrome P4501A (CYP1A), DT-diaphorase (DTD; EC 1.6.99.2), aldehyde dehydrogenase (ADH; EC 1.2.1.3), glutathione S-transferase (GST; EC 2.5.1.18) and UDP-glucuronyltransferase (UDPGT). DTD was assayed as dicoumarol-inhibitable menadione reductase, dicoumarol-inhibitable dichlorophenolindophenol (DCPIP) reductase and resorufin reductase activities; and ADH as benzaldehyde dehydrogenase (BADH) and propionaldehyde dehydrogenase (PADH) activities. The responses varied considerably with enzyme, species and treatment. CYP1A-dependent 7-ethoxyresorufin O-deethylase (EROD) activity was markedly inducible, more so in BNF-treated O. mykiss (maximal 284-fold increase after 2 days) than the 3MC-treated species (maximal 24–30-fold). Modest increases (up to 2-fold) and/or inhibition were seen in UDPGT and GST activities. DTD activities increased about 2-fold after 8 to 15 days in BNF-treated O. mykiss, but were not elevated in the other species. Changes in dicoumarol-inhibitable DCPIP and menadione reductase activities were more similar than resorufin reductase activity. No marked increases were evident in BADH, PADH, superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione peroxidase (GPX; EC 1.11.1.9) activities in any species. Overall, some co-induction of CYP1A and DTD, over different time-courses, was indicated for BNF-treated O. mykiss.

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles.

Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

Studies on bacterial nitroreductases. Enzymes involved in reduction of aromatic nitro compound in Escherichia coli.

Enzymes involved in reduction of methyl p-nitrobenzoate in Escherichia coli B/r were oxygen-insensitive and precipitated between 30 and 60% ammonium sulfate saturation from cell-free extracts of the strain. The reductases were resolved by DEAE-cellulose column chromatography into three enzymes, NADH-linked, NAD(P)H-linked and NADPH-linked ones. These enzymes were flavoprotein which could be inactivated by dialysis against 1 M potassium bromide and could be reactivated by FMN. The NADH-linked and NAD(P)H-linked reductases were sensitive to dicumarol and exhibited menadione reductase activities. Aromatic nitro compounds with electron-withdrawing p-substituents were easily reduced by the NAD(P)H-linked reductase.

Studies on the enzymatic reduction of C-nitroso compounds. V. Molecular properties of porcine heart C-nitrosoreductase and identity of this enzyme with NAD(P)H dehydrogenase.

NAD(P)H-dependent C-nitrosoreductase of porcine heart cytosol was purified 12,000-fold in the presence of NADH with an overall yield of 2.2%. The purification procedure included ammonium sulfate fractionation, gel filtration with Sephadex G-100, ion-exchange chromatography on DEAE-Sephadex A-50, hydrophobic chromatography on Octyl-Sepharose CL-4B, and gel filtration with Sephadex G-200. The purity of the preparation was approximately 90% and the molecular weight of the enzyme estimated by gel filtration was about 60,000. The purified enzyme was composed of two molecular forms, nitrosoreductases 1 and 2, having isoelectric points of 8.45 and 8.6, respectively. A significant amount of zinc was found in the preparation by X-ray fluorescence analysis. The enzyme as it was prepared was colorless, but, after oxidation with p-nitrosophenol followed by gel filtration in the absence of NADH, it showed the absorption spectrum of a flavoprotein. Spectral data indicated the presence of 1 mol of flavin per mol of the enzyme. The molecular turnover number was calculated to be 10,000 nmol p-nitrosophenol reduced to p-aminophenol per min per nmol enzyme at pH 5.8 and 22 degrees C. The activity was inhibited by p-chloromercuribenzoate by 50% at a concentration of 3 x 10(-5) M. Besides the nitrosoreductase activity, the purified preparation showed NAD(P)H-dependent menadione reductase activity. The activities were both strongly inhibited by dicumarol and markedly activated by serum albumin and by Tween 20. These results indicate the probable identity of this enzyme with soluble NAD(P)H dehydrogenase (quinone) [EC 1.6.99.2].

Proton translocation in cytochrome-deficient mutants of Escherichia coli

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.

Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: III. MECHANISM OF THE EFFECT OF SALT ON MENADIONE REDUCTASE

Abstract Halobacterium cutirubrum extracts exhibit no menadione reductase activity in the absence of salt; activity increases with salt concentration to a maximum at 2 m or higher concentrations of sodium chloride. The enzyme activity lost at low salt concentrations can be restored by adding 2 m NaCl. In addition to this reversible loss of activity, the enzyme also exhibits a time-dependent irreversible inactivation at salt concentrations below 2 m. The effects of salt on the initial enzyme activity and on the irreversible inactivation have a reciprocal relationship to one another. A mechanism for the salt effect is proposed, on the basis of kinetic data, whereby the active, stable form of the enzyme is in a salt-dependent equilibrium with an inactive, unstable form. The effect of NaCl, according to this model, is not to prevent the irreversible denaturation of the enzyme but rather to shift the equilibrium in favor of the active, stable form. The pH dependence of the inactivation rate constant and the reversibility of the pH effect indicate that there is an acid-base equilibrium, with pK = 9.3, between a less and a more stable intermediate form of the enzyme. Low concentrations of MgCl2, CaCl2, and some polyamines also have activating and protecting effects on menadione reductase. The mode of action of these substances is shown to be unlike that of NaCl but to consist in the alteration of the intermediate form of the enzyme in such a way as to make it more stable and partially active.

REDUCED NICOTINAMIDE–ADENINE DINUCLEOTIDE OXIDATION IN ESCHERICHIA COLI PARTICLES: III. CELLULAR LOCATION OF MENADIONE REDUCTASE AND ATPase ACTIVITIES

Sonic extracts of Escherichia coli were fractionated to membrane-wall, small-particle, and soluble fractions. Menadione reductase and ATPase activities were found in all fractions. The menadione reductase activity of the soluble fraction was resolved into three enzymes. Enzymes with properties similar to two of these could be released from the particulate fraction by treatment with sodium dodecyl sulfate or chelating agents. The major ATPase of the particulate fractions was solubilized by detergent. Osmotic shock experiments suggested that it was located outside the plasma membrane in the intact cell. It differed from the ATPase of the soluble fraction. An enzyme resembling the latter ATPase was solubilized from the small-particle fraction by chelating agents and could be rebound in the presence of magnesium ions. Solubilized menadione reductase was not rebound to particulate fractions under these conditions.

Diaphorases from Aerobacter aerogenes.

Bernofsky, Carl (The University of Kansas, Kansas City), and Russell C. Mills. Diaphorases from Aerobacter aerogenes. J. Bacteriol. 92:1404-1414. 1966.-Five enzymes which catalyze the reduction of 2,6-dichlorophenol-indophenol by reduced nicotinamide adenine dinucleotide (NADH(2)) have been separated from sonic extracts of Aerobacter aerogenes B199 by diethylaminoethyl (DEAE) cellulose chromatography. Three major chromatographic fractions (enzymes I, II, and III) account for most of the activity in the extract. Of the two minor fractions, one is associated with cytochrome b(1). The other is extremely labile, and was not studied further. The chromatographed diaphorases appear to have a specific requirement for flavin mononucleotide. They are also readily inactivated by dilution; however, this can be prevented by a combination of phosphate buffer, bovine serum albumin, and flavin mononucleotide. The different enzymes are clearly distinguishable by their activities with NADH(2) and reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) in the presence of various electron acceptors (2,6-dichlorophenol-indophenol, ferricyanide, menadione, and cytochrome c), and by their responses to inhibitors (amobarbital, antimycin A, Atabrine, p-chloromercuribenzenesulfonate, dicumarol, and 2,4-dinitrophenol). With 2,6-dichlorophenol-indophenol as acceptor, enzymes I, II, and III have comparable activities with either NADH(2) or NADPH(2). With menadione and ferricyanide as acceptors, enzymes II and III exhibit very high, NADH(2)-specific activities. When cytochrome c is the acceptor, however, enzyme III shows greater activity with NADPH(2) as the electron donor. Ferricyanide is the most active acceptor for the cytochrome b(1)-containing fraction. Coenzyme Q(6) does not appear to serve as an acceptor. All the diaphorases, with the exception of that in the cytochrome b(1)-containing fraction, are inhibited by p-chloromercuribenzenesulfonate. Amobarbital is relatively ineffective and inhibits only the indophenol reductase activity of enzyme I. The menadione reductase activity of enzymes I, and II, and the diaphorases in the cytochrome b(1)-containing fraction are strongly inhibited by antimycin A, 2,4-dinitrophenol, dicumarol, and Atabrine. However, the menadione reductase activity of enzyme III is affected only by the last three of these inhibitors. The diaphorases in sonic-treated extracts do not appear to be associated with a particulate fraction.