• Korean Journal of Microbiology
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• v.39 no.4
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• pp.223-229
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• 2003

The purpose of this work was to perform the characterization of NAD(P)H-nitroreductase isolated from Stenotrophomonas sp. OK-5 capable of degrading 2,4,6-trinitrotoluene (TNT). Initially, NADP(H)-nitroreductase by a series of purification processes including ammonium sulfate precipitation, DEAE-sepharose, andQ-sepharose was prepared. From samples harvested from fraction collector, three different fractions (I, II & III)having the enzyme activity of NAD(P)H-itroreductase were detected. Specific activities of three fractions I, II,and III of NAD(P)H-nitroreductase were determined to approximately 5.06 unit/mg, 4.95 unit/mg and 4.86 unit/mg, and concentrated to 10.5, 9.8, and 8.9-fold compared to crude extract, respectively. Among these three fractions,the fraction I of NAD(P)H-nitroreductase demonstrated the highest specific activity in this experiment. Several factors affecting on the enzyme activity of NAD(P)H-nitroreductase (fractions I, II & III) were investigated.The optimum temperature of all NAD(P)H-nitroreductase (fractions I, II & III) was 30oC, and the optimal pH was approximately 7.5. Metal ions such as Ag+, Cu2+, Hg2+ inhibited approximately 80% enzyme activity of all NAD(P)H-nitroreductase, and the enzyme activities were decreased about 30-40% inhibition in the presence of Mn2+ or Ca2+. However, Fe3+ showed stimulatory effect on the enzyme activity. The molecular weights of NAD(P)H-nitroreductase (fractions I, II & III) were measured about 27 kDa on the SDS-PAGE.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.1
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• pp.15-19
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• 2002

The role of vascular NAD(P)H oxidase in subarachnoid hemorrhage (SAH)-induced vasospasm in the basilar artery was examined in a rat model. Arterial vasospasm characterized by increased wall thickness and decreased lumen size was observed at 5 to 7 days after $2^{nd}$ injection of blood into cisterna magna, and these changes were significantly ameliorated by pretreatment of diphenyleneiodonium $(DPI,\;25\;{\mu}l\;of\;100\;{\mu}M),$ an inhibitor of NAD(P)H oxidase. To determine the time course of changes in the vascular NAD(P)H oxidase activity, cerebral vasculature was isolated at different time intervals from 12 hrs to 14 days after injection of autologous blood. At 24 hrs after the second injection of blood, the NAD(P)H oxidase activity was markedly increased with an enhanced membrane translocation of p47phox, but by 48 hours both the enzyme activity and p47phox translocation regained normal values, and were remained unchanged up to 14 days after SAH. However, no significant changes in the expression of p22phox mRNA was observed throughout the experiments. These findings suggest that the activation of NAD(P)H oxidase by which assembly of the oxidase components enhanced and subsequent production of superoxide in the early stages of SAH might contribute to the delayed cerebral vasospasm in SAH rats.

• BMB Reports
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• v.32 no.2
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• pp.127-132
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• 1999

The NAD(P)H-quinone oxidoreductase (EC 1. 6. 99. 2) was purified from S. cerevisiae. The native molecular weight of the enzyme is approximately 111 kDa and is composed of five identical subunits with molecular weights of 22 kDa each. The optimum pH of the enzyme is pH 6.0 with 1,4-benzoquinone as a substrate. The apparent $k_m$ for 1,4-benzoquinone and 1,4- naphthoquinone are 1.3 mM and $14.3\;{\mu}M$, respectively. Its activity is greatly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, nitrofurantoin, dicumarol, and Cibacron blue 3GA. The purified NAD(P)H-quinone oxidoreductase was found capable of reducing aromatic nitroso compounds as well as a variety of quinones, and can utilize either NADH or NADPH as a source of reducing equivalents. The nitroso reductase activity of the purified NAD(P)H-quinone oxidoreductase is strongly inhibited by dicumarol.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1346-1351
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• 2018

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes $NAD(P)^+-dependent$ glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

• BMB Reports
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• v.32 no.4
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• pp.321-325
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• 1999

NAD(P)H-quinone oxidoreductase (EC 1. 6. 99. 2) was purified form S. cerevisiae. The enzyme readily reduced 2,6-dichlorophenolindophenol, a quinonoid redox dye, as well as substituted benzo- and naphthoquinones, and could accept electrons from either NADH or NADPH. The purified NAD(P)H-quinone oxidoreductase turned out to be capable of reducing nitrosoarenes as well as a variety of quinones. A chemical-trapping technique using 4-chloro-1-naphthol was used to show that the N,N-dimethyl-p-benzoquinonediiminium cation was produced in the reduction of 4-nitroso-N,N-dimethylaniline catalyzed by NAD(P)H-quinone oxidoreductase.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.2
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• pp.103-109
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• 2003

The cellular mechanisms that contribute to the acceleration of atherosclerosis in diabetes are poorly understood. Therefore, the potential mechanisms involved in the diabetes-dependent increase in vascular smooth muscle cell (VSMC) proliferation was investigated. Using primary culture of VSMC from streptozotocin-induced diabetic rat aorta, cell proliferation assay showed two-fold increase in cell number accompanied with enhanced superoxide generation compared to normal VSMC, 2 days after plating. Both the increased superoxide production and cell proliferation in diabetic VSMC were significantly attenuated by not only tiron (1 mM), a superoxide scavenger, but also by diphenyleneiodonium (DPI; $10{\mu}M$), an NAD(P)H oxidase inhibitor. NAD(P)H oxidase activity in diabetic VSMC was significantly higher than that in control cell, accompanied with increased mRNA expression of p22phox, a membrane subunit of oxidase. Furthermore, inhibition of p22phox expression by transfection of antisense p22phox oligonucleotides into diabetic VSMC resulted in a decrease in superoxide production, which was accompanied by a significant inhibition of cell proliferation. Based on these results, it is suggested that diabetes-associated increase in NAD(P)H oxidase activity via enhanced expression of p22phox contributes to augmented VSMC proliferation in diabetic rats.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.1
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• pp.31-36
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• 2007

To elucidate a potential molecular link between diabetes and atherosclerosis, we investigated the role of Janus tyrosine kinase(JAK) for NAD(P)H oxidase-derived superoxide generation in the enhanced proliferative capacity of vascular smooth muscle cells(VSMC) of Otsuka Long-Evans Tokushima Fatty(OLETF) rat, an animal model of type 2 diabetes. An enhanced proliferative response to 10% fetal bovine serum(FBS) and superoxide generation with an increased NAD(P)H oxidase activity were observed in diabetic(OLETF) VSMC. Both the enhanced proliferation and superoxide generation in diabetic VSMC were significantly attenuated by AG490, JAK2 inhibitor, and PP2, Src kinase inhibitor. Tyrosine phosphorylation of proteins in diabetic VSMC, especially JAK2, was increased compared to control VSMC. Furthermore, the enhanced NAD(P)H oxidase activity in diabetic VSMC was significantly attenuated by AG490 in a dose-dependent manner. Together, these results indicate that the signal pathway which leads to diabetes-associated activation of Src kinase/JAK is critically involved in the diabetic VSMC proliferation through NAD(P)H oxidase activation and superoxide generation.

• Molecules and Cells
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• v.25 no.2
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• pp.158-162
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• 2008

Recent molecular genetics studies have revealed that cyclic electron transport around photosystem I is essential for normal photosynthesis and growth of plants. Chloroplastic NAD(P)H dehydorgenase (NDH) complex, a homologue of the complex I in respiratory electron transport, is involved in one of two cyclic pathways. Recent studies on the function and structure of the NDH complex are reviewed.

• KSBB Journal
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• v.4 no.3
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• pp.229-234
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• 1989

NAD+ analogs, 8-( 6-aminohexyl) aminonicotinamide adenine dinucleotide and N6-[(6- aminohewl)-carbamoylmethyl]- NAD+, were imobilized on bovine caseins by the action of hansglutaminase. It appears that NAD+ analogs bind with $\alpha$S1-and $\beta$-caseins through formation of the r-glutamylamine bond between the amino groups attached to the hexyl chains in NAD+ analogs and the glutaminyl residues in caseins. The NAD+ analogs immobilized on the caseins were enzymatically reducible by alcohol dehydrogenase. $\beta$-Casein was more useful carrier than the $\alpha$S1-casein and 8-substituted NAD+ analog was more effective than N6-substituted one in immobilization. Michaelis constant of 8-substituted NAD+ analog immobilized on $\beta$-casein in alcohol dehydrogenase reaction was similar to that of free from of NAD+ and that of NAD+ analog. Immobilized NAD+ was much more stable at alkaline pH than free NAD+ and its analog while maximum velocity was reduced to 31% of the free NAD+ analog. The coenzyme casein conjugated was recovered almost completely in casein precipitated by calcium.

• Journal of the Korean Chemical Society
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• v.23 no.4
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• pp.248-258
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• 1979

Glucose 6-phosphate dehydrogenase of Leuconostoc mesenteroides which was purifid by an affinity chromatography was studied on the characterization, kinetics and chemical modification. The apparent molecular weight of the enzyme was 112,000 by the gel filtration method of Sephadex G-200 column. The optimum temperature of $NAD^+$-linked reation was 50$^{circ}C$ and the activation energy and the heat of inactivation were 8.36 kcal/mole and -58.2kcal/mole, respectively. The steady state kinetic study showed KG6P, Kemp, and CX KNADP to be 76.9 PM, 7.46${\mu}M$ and 7.14 ${\mu}M$, respectively, and KGGP, KNAD,and aKNm to be 53.7${\mu}M$, 115.2${\mu}M$ and 702.2${\mu}M$ for the $NAD^+$-linked reaction at pH 7.8, optimum pH. The pH dependent kinetic constants suggested that the two ionizing groups whose pKa is 7.2 .and pKb is 9.0-9.6 were involved in the enzyme-substrate interaction. Evidence by photooxidation and carboxymethylation of the enzyme suggested that the imidazole group of histidine with pKa group may participate in the catalytic site.