• 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.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.35-42
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• 2010

Many studies propose that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. With a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1) as the gene delivery method, we were able to attain high transduction efficiencies even in the human epithelial cervical cancer cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. Using a rAAV-NDI1, we demonstrated that the Ndi1 enzyme is successfully expressed in HeLa cells. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced HeLa cells were not affected by rotenone which is inhibitor of complex I, but was inhibited by flavone and antimycin A. The NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. In particular, in the NDI1-transduced cells, the yeast enzyme becomes integrated into the human respiratory chain. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.427-434
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• 2011

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber's hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

• Journal of Microbiology
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• v.45 no.4
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• pp.333-338
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• 2007

Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between $20-40^{\circ}C$, with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by $CuSO_4,\;HgCl_2,\;MgSO_4,\;MnSO_4,\;AgNO_3$, dicumarol, KCN, $NaN_3$, and EDTA. Its $K_m\;and\;V_{max}$ with NADH as an electron donor were $23{\mu}M\;and\;101mM/mg$ per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.6
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• pp.331-334
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• 2002

Culture conductivity and on-line NADH fluorescence were used to measure cellular growth in plant cell suspension cultures of Podophyllum hexandrum. An inverse correlation between dry cell weight and medium conductivity was observed during shake flask cultivation. A linear relationship between dry cell weight and culture NADH fluorescence was obtained during the exponential phase of batch cultivation In a bioreactor under the pH stat (pH 6) conditions. It was observed that conductivity measurement were suitable for biomass characterisation under highly dynamic uncontrolled shake flask cultivation conditions. However, if the acid/alkali feeding is done for pH control the conductivity measurement could not be applied. On the other hand the NADH fluorescence measurement allowed online-in situ biomass monitoring of rather heterogenous plant cell suspension cultures in bioreactor even under the most desirable pH stat conditions.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.206.1-206
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• 1996

No Abstract, See Full Text

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.7-14
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• 1998

To study the oxygen tolerance mechanism of bifidobacteria, we have studied the growth of cells, the activities of the enzymes which were related with oxygen, such as catalase, superoxide dismutase(SOD), NADH oxidase, and NADH peroxidase, and cellular fatty acid compositions of Bifidobacterium adolescentis and B. longum under anaerobic and aerated (microaerobic and aerobic) conditions. B. longum grew relatively well under the microaerobic conditions, whereas the growth of B. adolescentis was inhibited under the same aerated conditions. B. adolescentis had extremely low level of NADH oxidative enzymes while B. longum had the relatively high level of NADH oxidative enzymes, whose activities were dramatically increased from 3.7 to 11.4 times by microaerobic condition but not in B. adolescentis. The activity of SOD was unexpectedly high in B. adolescentis compared with in B. longum under anaerobic and aerated conditions. The activities of catalase were not detected in all samples tested in this study. We also found that normal $C_{l6:0}$ and $C_{18:1}$ were the major fatty acids in B. adolescentis and B. longum under anaerobic and aerated conditions. 2.2-14.1% $C_{l9:0}$ cyclo fatty acid was detected only in B. longum and the fatty acid was increased by the addition of the aeration. The $C_{l9:0}$ cyclic fatty acid was identified as a cis 9, 10-methylene octadecanoic acid, which was different from lactobacillic acid in the cyclized site. 6.6%-24.6% of dimethyl acetals (DMA) which came from plasmalogen were observed in the B. adolescentis and B. longum grown under anaerobic condition, and the components were notably decreased in the cells grown under the aerated conditions. It is believed that NADH oxidative enzymes play an important role to detoxify oxygen metabolites of Bifidobacteriurn spp. under anaerobic and microaerobic conditions. Independently from oxidative enzymes, it seems that oxygen stress may induce the change of the level of cellular fatty acids showing an increase of $C_{l9:0}$ cyclo in B. longum and a decrease of $C_{l8:1}$ of plasmalogen in B. longum and B. adolescentis to adapt in environment.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.160.3-161
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• 2003

Aldehyde and active form of free oxygen produced in alcohol metabolism in liver are the cause of liver cell damage. The main system of alcohol metabolism is composed of alcohol dehydrogenase(ADH), aldehyde dehydrogenase(ALDH) and cytochrome P4502E1. Alcohol dehydrogenase is reversible in alcohol metabolism. To block the backward reaction and enhance alcohol oxidation, acetaldehyde trapping agents were assayed. The assay was carried out by measuring decreasing NADH at 340nm, using acetaldcehyde and NADH as substrate and coenzyme respectively. (omitted)

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.362-367
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• 2009

In recombinant strains, many proteins and enzymes are expressed as inactive and insoluble inclusion bodies. For soluble expression of an active form of StyB, an NADH-flavin oxidoreductase, several recombinant Escherichia coli strains were developed and tested. Among them, strain BL21(DE3)pLysS effectively produced an active and soluble form of StyB as about 9% of the total protein content, when cultivated at $20^{\circ}C$ with 0.5 mM IPTG. The solubly expressed StyB has the highest oxidoreductase activity at pH 6.5-7.5 and $37^{\circ}C$. Substrate dependence profiles of the StyB-catalyzed reaction showed that the maximum specific activity($V_m$) and half saturation constant($K_m$) were $1,867{\pm}148\;U/mg$ protein and $51.6{\pm}11{\mu}M$ for NADH, and $1,274{\pm}34\;U/mg$ protein and $8.2{\pm}1.2{\mu}M$ for FAD, respectively. This indicates that solubly produced StyB has 6- to 9-fold higher oxidoreductase activities than the in vitro refolded StyB from inclusion bodies.

• Korean Journal of Medicinal Crop Science
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• v.12 no.2
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• pp.113-117
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• 2004

The experiment was conducted to evaluate the effects of medicinal plants on ethanol-metabolism. Sprague Dawley rats divided into 6 groups (n=8), fed with 10% ethanol and diets supplemented with each 1% of four plant extracts, ${\alpha}-tocopherol$ (as positive control) and fiber (as negative control) for 4 weeks. Group supplemented with plant extract of Ulmus davidiana showed the most high value (322 nM NADH/min/mg protein) in alcohol dehydrogenase (ADH) activity among the experimented groups $(144{\sim}312\;nM\;NADH/min/mg\;protein)$ at p<0.05. Groups fed with Lagerstroemia indica and Zelkova serrata extract-supplemented diets indicated high activity in aldehyde dehydrogenase (ALDH, 16.7 & 12.3 M NADH/min/mg protein), which were comparatively lower than 20.1 M NADH/min/mg protein of ${\alpha}-tocopherol$ fed group. All of the groups fed with plant extracts indicated very low GPT activities $(13.9{\sim}17.3\;IU/l)$ compared to those (146.1 & 128.6 IU/l) fed with ${\alpha}-tocopherol$ and fiber at p<0.05. From these results, it is suggested that Lagerstroemia indica have a potent ethanol-metabolizing activity.