• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.914-918
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• 2004

This study was carried out to analyze the metabolic flux of W. kimchii sk10 on pyruvate and ethanol as a carbon source. The sk10 grown on ethanol produced acetate under aerobic conditions rather than under anaerobic conditions. The lactate and acetate were produced on ethanol plus pyruvate by the sk10 grown under aerobic and anaerobic conditions, respectively. The resting cell of sk10 produced 99.1 mM acetate and 17.3 mM lactate under aerobic conditions and 51.1 mM acetate and 62.4 mM lactate under anaerobic conditions from ethanol plus pyruvate, respectively. This result is thought to be due to the difference in the $NADH/NAD^+$ ratio depending on the growth conditions. The 11-fold overproduction of NADH peroxidase results in a low $NADH/NAD^+$ratio under aerobic growth conditions. At the low $NADH/NAD^+$ ratio, the metabolic flux of pyruvate toward lactate has to be shifted to a flux toward acetate without NADH oxidation to $NAD^+$, and ethanol oxidation to acetate coupled to $NAD^+$ reduction to NADH has to be activated.

• Nutritional Sciences
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• v.6 no.4
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• pp.189-194
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• 2003

Most of the ethyl alcohol consumed by humans is oxidized to acetaldehyde in the liver by the cytoplasmic alcohol dehydrogenase (ADH) system. For this ADH-catalyzed oxidation of alcohol, $NAD^+$ is required as the coenzyme and $NAD^+$becomes reduced to NADH. As the $NAD^+$becomes depleted and NADH accumulates, alcohol oxidation is reduced. For continued alcohol oxidation, the accumulated NADH must be quickly reoxidized to $NAD^+$, and it is this reoxidation of NADH to $NAD^+$that is known to be the rate-limiting step in the overall oxidation rate of alcohol The reoxidation of NADH to $NAD^+$is catalyzed by lactate dehydrogenase in the cytoplasm of hepatocytes, with pyruvate being utilized as the substrate. The pyruvate may be supplied from alanine as a result of amino acid metabolism via the urea cycle. Also, glutamine is thought to help with the supply of pyruvate indirectly, and to activate the urea cycle by producing $NH_3$. Thus, in the present study, we have examined the effects of alanine and glutamine on the alcohol oxidation rate. We utilized isolated perfused liver tissue in a system where media containing alanine and glutamine was circulated. Our results showed that when alanine (5.0mM) was added to the glucose-free infusion media, the alcohol oxidation rate was increased by 130%. Furthermore, when both glutamine and alanine were added together to the infusion media, the alcohol oxidation rate increased by as much as 190%, and the rate of urea nitrogen production increased by up to 200%. The addition of glutamine (5.0mM) alone to the infusion media did not accelerate the alcohol oxidation rate. The increases in the rates of alcohol oxidation and urea nitrogen production through the addition of alanine and glutamine indicate that these amino acids have contributed to the enhanced supply of pyruvate through the urea cycle. Based on these results, it is concluded that the dietary supplementation of alanine and glutamine could contribute to increased alcohol detoxification through the urea cycle, by enhancing the supply of pyruvate and $NAD^+$to ensure accelerated rates of alcohol oxidation.

• Bulletin of the Korean Chemical Society
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• v.25 no.6
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• pp.786-790
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• 2004

2-Aminoethanethiol (aet) has been used to make self-assembled monolayer (SAMs) on gold electrodes, which are subsequently modified with 3,4-dihydroxy phenylalanine (dpa). Such modified electrodes having various types of Au/aet-dpa were employed in the electrocatalytic oxidation of NADH. The purpose of this study to characterize the responses of such modified electrodes in terms of the immobilization procedure, pH of the solution and applied potential. The reaction of the surface immobilized dpa with NADH was studied using the rotating disk electrode technique and a value of $2.2{\times}10^4M^{-1}s^{-1}$ was obtained for the second-order rate constant in 0.1 M Tris/$NO_3^-$buffer (pH=8.0). The hydration behavior of the films was characterized by quartz crystal microbalance. When used as a NADH sensor, the Au/aet-dpa electrode exhibited good sensitivity and an excellent correlation (r ${\geq}$ 0.99) for NADH concentration which extended to $3.8{\times}10^{-3}$ M.

• Bulletin of the Korean Chemical Society
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• v.33 no.12
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• pp.4211-4214
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• 2012

• BMB Reports
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• v.40 no.1
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• pp.53-57
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• 2007

The enzymatic properties of NADH:quinone oxidoreductase were examined in Triton X-100 extracts of Bacillus cereus membranes by using the artificial electron acceptors ubiquinone-1 and menadione. Membranes were prepared from B. cereus KCTC 3674 grown aerobically on a complex medium and oxidized with NADH exclusively, whereas deamino-NADH was determined to be poorly oxidized. The NADH oxidase activity was lost completely by solubilization of the membranes with Triton X-100. However, by using the artificial electron acceptors ubiquinone-1 and menadione, NADH oxidation could be observed. The activities of NADH:ubiquinone-1 and NADH:menadione oxidoreductase were enhanced approximately 8-fold and 4-fold, respectively, from the Triton X-100 extracted membranes. The maximum activity of FAD-dependent NADH:ubiquinone-1 oxidoreductase was obtained at about pH 6.0 in the presence of 0.1M NaCl, while the maximum activity of FAD-dependent NADH:menadione oxidoreductase was obtained at about pH 8.0 in the presence of 0.1M NaCl. The activities of the NADH:ubiquinone-1 and NADH:menadione oxidoreductase were very resistant to such respiratory chain inhibitors as rotenone, capsaicin, and $AgNO_3$, whereas these activities were sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Based on these results, we suggest that the aerobic respiratory chain-linked NADH oxidase system of B. cereus KCTC 3674 possesses an HQNO-sensitive NADH:quinone oxidoreductase that lacks an energy coupling site containing FAD as a cofactor.

• The Korean Journal of Pharmacology
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• v.20 no.1 s.34
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• pp.1-11
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• 1984

The present study was performed to determine oxygen metabolites involved in cell-free MPO/$H_2O_2/Cl^-$ system by observing the effects of their scavengers on NADH oxidation and ethylene production from methional by the action of MPO prepared from human leukocytes. It was clearly demonstrated that NADH was oxidized by the cell-free MPO/$H_2O_2/Cl^-$ system as evidenced by complete inhibition of the oxidation of the substrate in the presence of eiher azide or catalase, or by omitting $Cl^-$. The MPO-mediated oxidation of NADH was completely abolished by a $^1O_2$ quencher, DABCO but not by $OH{\cdot}$ scavengers, mannitol, benzoate, formate and methanol. In ethylene assay, no ethylene was detected from methional in the MPO/$H_2O_2/Cl^-$ system with evident production of the gas by xanthine-oxidase and $Cu^{++}-H_2O_2$ systems which are suggested to generate $OH{\cdot}$. From the results obtained, it is concluded that $^1O_2$ is a major mediator with exclusion of $OH{\cdot}$ involvement in the cell-free MPO-mediated oxidation.

• BMB Reports
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• v.37 no.4
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• pp.416-421
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• 2004

The antioxidant activities of NADH and of its analogue, 1,4-dihydro-2,6-dimethyl-3,5-dicarbethoxy-pyridine ($PyH_2$), were evaluated in vitro. NADH was found to be oxidized by the peroxyl radical derived from 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH) decomposition, in a pH-dependent manner. Both NADH and $PyH_2$ inhibited the peroxidation of egg yolk lecithin (EYL) liposomes, although $PyH_2$ was more effective than NADH when 2,2'-azobis-4-methoxy-2,4-dimethyl-valeronitrile (methoxy-AMVN) was employed to induce EYL liposome peroxidation. The antioxidant activities of NADH and $PyH_2$ were also evaluated by measuring their influences on 1,3-diphenylisobenzofuran (DPBF) fluorescence decay in the presence of peroxyl radicals. NADH and $PyH_2$ were much more effective at inhibiting DPBF quenching in Triton X-100 micelles than in liposomes. These results indicate that NADH can inhibit lipid peroxidation despite being hydrophilic. Nevertheless, membrane penetration is an important factor and limits its antioxidant activity.

• Journal of the Korean Electrochemical Society
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• v.10 no.1
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• pp.1-6
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• 2007

Mercaptopropionic acid(mpa) has been used to make self-assembled monolayer(SAMs) on the surface of graphite electrode incorporating gold nano particles, which are subsequently modified with dopamine(dopa). Such modified electrodes haying types of Gr(Au)/mpa-dopa were employed in the electrocatalytic oxidation of NADH. The responses of such modified electrodes were studied in terms of electron transfer kinetics and reaction procedure in the reaction. The reaction of the surface immobilized dopa with NADH was studied using the rotating disk electrode technique and a value of $5.06{\times}10^5M^{-1}s^{-1}$ was obtained for the second-order rate constant in 0.1 M phosphate buffer(pH=7.0), which was a $EC_{cat}$ and kinetic controlled procedure. But, the modified electrodes were diffusion controlled reaction having $4.64{\times}10^{-4}cm^2s^{-1}$ of the coefficient within $10^{-3}s$ after starting the reaction.

• Bulletin of the Korean Chemical Society
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• v.30 no.12
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• pp.2984-2988
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• 2009

A gene (PH0311) encoding a hypothetical protein from the genome sequence data of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and over-expressed in Escherichia coli. The purified recombinant protein was found to possess FAD-dependent NADH oxidase activity, although it lacked sequence homology to any other known general NADH oxidase family. The product of the PH0311 gene was thus designated PhNOX (NADH oxidase from Pyrococcus horikoshii), with an estimated molecular weight of 84 kDa by gel filtration and 22 kDa by SDS-PAGE, indicating it to be a homotetramer of 22 kDa subunits. PhNOX catalyzed the oxidation of reduced ${\beta}$-NADH with subsequent formation of $H_2O_2$ in the presence of FAD as a cofactor, but not ${\alpha}$-NADH, ${\alpha}$-NADPH, or ${\beta}$-NADPH. PhNOX showed high affinity for ${\beta}$-NADH with a Km value of 3.70 ${\mu}$M and exhibited optimum activity at pH 8.0 and 95$^{\circ}C$ as it is highly stable against high temperature.

• Journal of Sensor Science and Technology
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• v.32 no.6
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• pp.412-417
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• 2023

There is increasing interest in the rapid and highly sensitive monitoring of cell viability in biological and toxicological research. Conventional methods depend on optical assays using Water Soluble Tetrazolium-8 (WST-8) or 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, which requires a large volume of samples and special instruments, necessitating shipment of clinical samples to laboratories. This paper reports on the development of a rapid and sensitive electrochemical (EC) sensor using screen printed electrode (SPE) and surface modification using 4'-mercapto-N-phenylquinone diamine (4'-NPQD), as double electron mediators, for monitoring cell viability via the measurement of nicotinamide adenine dinucleotide (NADH). We used the sensor to observe the viability of MCF-7 and doxorubicin (Dox)-treated cells. The oxidation current of NADH was measured via chronoamperometry (CA), and the EC results showed a good linear relationship when compared with NADH quantification using WST-8 assay. The analysis time was only 10 s and limit of detection (LOD) of NADH was 1.78 µM. Our EC method has the potential to replace conventional WST assays for cell viability and cytotoxicity experiments.