• BMB Reports
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• 제32권2호
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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.

• Animal cells and systems
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• 제4권2호
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• pp.141-144
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• 2000

Effects of deamido-$\textrm{NAD}^{+}$on self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) was investigated. The self-splicing was not affected by deamido-$\textrm{NAD}^{+}$- at concentrations up to 2 mM. However, it began to decrease at 5 mM and the formation of splicing products such as the linear intron, intron-exon 2 and exon 1-exon 2, was slightly reduced. At 20 mM the self-splicing activity was almost completely abolished. This analog of the coenzyme $\textrm{NAD}^{+}$- inhibits the self-splicing of td intron RNA although it does not possess a guanidine group in its structure. The analysis of inhibitory concentrations and structural examination suggests that the key structural features of deamido-$\textrm{NAD}^{+}$ responsible for the inhibition of splicing may be the ADP-ribose moiety.

• 생명과학회지
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• 제8권2호
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• pp.181-188
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• 1998

두 종류의 ADP-riboyltransferase(Yac-1과Yac-2)가 최근 mouse의 임파구로부터 cloning되어 그 특성이 규명되어졌다. Yac-1효소는 ADP-ribosyltransferase활성을 보여주나, 대조적으로, Yac-2는 상당한 양의 NAD glycohydrolase 활성도 소유하고` 있으며 이는 NDA를 우선적으로 가수분해 할 수 있다는 사실을 반영한다. Yac-2는 보존된 두 glutamate에 인접한 위치인 219번에 또 다른 glutamate를 소유하고 있다. 효소 활성에 대한 Glu-219의 효과를 알아보기 위해 두 단계의 재조합 중합효소 연쇄 반응 방법에 의해 Glu-219가 glutamate (E219Q)혹은 alanine(E219A)으로 치환되었다. Gln혹은 Ala으로의 치환결과, ADP-ribosyltransferase활성은 56% (E219Q)혹은 66% *E219A)로 감소하였다. Yac-2단백질의 NAD glycohydrolase 활성은 돌연변이에 의해 영향을 받지 않았다. 이러한 결과는 Yac-2효소의 Glu-219가 ADP-ribosyltransferase 활성에 중요한 역할을 하나, NDA glycohydrolase활성에는 관여하지 않음을 시사한다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 추계 학술대회
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• pp.181-188
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• 2000

The electrocatalytic reduction of NAD$^{＋}$ using diaphorase was studied. methyl viologen (MV$^{2＋}$) mediator between an electrode and the enzyme. Steady-state currents could be obtained under the conditions of slow scan rate, low MV$^{2＋}$concentration, and high NAD$^{＋}$ concentration as the electrode reaction was converted to an electrochemical-catalytic (EC') reaction. The biomecular rate constant for the reaction of the reduced methyl viologen with the oxidized diaphorase was estimated as 7.5$\times$10$^3$M$^{-1}$ s$^{-1}$ from the slope of the current versus [MV$^{2＋}$] plot. And the optimal concentrations of diaphorase, MV$^{2＋}$ and NAD$^{＋}$ in the mediated electrocatalytic reduction of NAD$^{＋}$ were decided by applying the cyclic voltammetry. The optimal concentrations of the species were obtained by finding the conditions which gave the highest and steady-state current at a gold-amalgam electrode. The highest and steady-state catalytic current was achieved under the conditions of 1.5 U/ml diaphorase, 0.2 mM MV$^{2＋}$, and 4.8 mM NAD$^{＋}$ at the scan rate of 2 mV s$^{-1}$ , suggesting that the rate of the electrocatalytic reation is the higest under the former conditions. The electrochemical procedure under the conditions of 1.5 U/ml diaphorase,0.2 mM MV$^{2＋}$, and 4.8 mM NAD$^{＋}$ was used favorably to drive an enzymatic reduction of pyruvate to D-lactate.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.510.1-510
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• 1986

Many enzymes require the participation of readily dissociable coenzymes as NAD for thir catalytic activities. The continuous utilization of the enzymes requires the retention and regeneration of the coenzymes. For this purpose, several kinds of macromolecular NAD derivatives have been prepared by covalently attaching NAD to watersoluble polymers. We have prepared poly (ethylene glycol)-bound NAD (PEG-NAD) by coupling N$\^$6/-(2-carboxyethyl)-NAD to one terminal of ${\gamma}$ $\omega$-diaminoly (ethylene glycol) (Mr 3000) with water-soluble carbodiimide. PED-NAD thus obtained has one NAD moiety located at a terminal of the linear, flexible and hydrophilic chain of poly (ethylene glycol). PED-NAD has good coenzyme activity for various dehydrogenases and is applicable in a continuous enzyme reactor. To use these macromolecular NAD derivatives in an enzyme reactor, it si necessary to understand the behavior of the system in which the reactions of dehydrogenases are coupled by the recycling of the NAD derivative. We investigated the kinetic properties of a continuous enzyme reactor containing lactate dehydrogenase, alcohol dehydrogenase and PEG-NAD. The steady-state behavior of the enzyme reactor is explained by a simple kinetic model.

• Journal of Applied Biological Chemistry
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• 제42권1호
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• pp.19-24
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• 1999

Plants have been shown to contains $Ca^{2+}$/calmodulin-stimulated GAD and NAD kinase. To test how calmodulin and calmodulin methylation affect the activation of GAD and NAD kinase, GAD and NAD kinase were partially purified from tobacco plants. GAD was also partially purified from E. coli transformed with a plasmid carrying a cloned tobacco GAD gene. We find that GAD from the transformed E. coli showed 60-fold $Ca^{2+}$/calmodulin-dependent activation. However, GAD from tobacco plants was stimulated only about 3.8-fold by the addition of calmodulin in the presence of calcium, suggesting high background activity of the enzyme was possibly due to bound endogenous tobacco calmodulin. There were no significant differences in the tobacco GAD activator properties between calmodulins. A monoclonal antibody against petunia GAD interacted strongly with both GAD from tobacco plants and GAD from cloned gene. NAD kinase from tobacco plants showed a complete $Ca^{2+}$/calmodulin dependency for activity. Unmethylated calmodulins activated GAD in a manner similar to methylated calmodulin. However, the maximum level of NAD kinase activation obtained with unmethylated calmodulins is approximately 4-fold higher than methylated calmodutins. These data suggested that endogenous tobacco calmodulin may interact more tightly with GAD than NAD kinase and that calmodulin methylation affects the activator properties of calmodulins for tobacco NAD kinase but not for GAD.

• Molecules and Cells
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• 제24권3호
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• pp.351-357
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• 2007

Regions (about 3.7-3.8 kb) of the mitochondrial genomes (rrnL-cox1) of two tardigrades, a heterotardigrade, Batillipes pennaki, and a eutardigrade, Pseudobiotus spinifer, were sequenced and characterized. The gene order in Batillipes was $\underline{rrnL}-\underline{V}-\underline{rrnS}-\underline{Q}-\underline{I}$-M-nad2-W-$\underline{C}-\underline{Y}$-cox1, and in Pseudobiotus it was $\underline{rrnL}-\underline{V}-\underline{rrnS}-\underline{Q}$-M-nad2-W-$\underline{C}-\underline{Y}$-cox1. With the exception of the trnI gene, the two tardigrade regions have the same gene content and order. Their gene orders are strikingly similar to that of the chelicerate Limulus polyphemus (rrnL-V-rrnS-CR-I-Q-M-nad2-W-C-Y-cox1), which is considered to be ancestral for arthropods. Although the tardigrades do not have a distinct control region (CR) within this segment, the trnI gene in Pseudobiotus is located between rrnL-trnL1 and trnL2-nad1, and the trnI gene in Batillipes is located between trnQ and trnM. In addition, the 106-bp region between trnQ and trnM in Batillipes not only contains two plausible trnI genes with opposite orientations, but also exhibits some CR-like characteristics. The mitochondrial gene arrangements of 183 other protostomes were compared. 60 (52.2%) of the 115 arthropods examined have the M-nad2-W-C-Y-cox1 arrangement, and 88 (76.5%) the M-nad2-W arrangement, as found in the tardigrades. In contrast, no such arrangement was seen in the 70 non-arthropod protostomes studied. These are the first non-sequence molecular data that support the close relationship of tardigrades and arthropods.

• 생명과학회지
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• 제8권5호
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• pp.486-491
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• 1998

mouse의 임파구로부터 두 종류의 ADP-ribosyltransferase (Yac-1과 Yac-2)가 클로닝되어 특성을 규명한 바 있다. Yac-2는 ADP-ribosyltransferase 활성 뿐 아니라 높은 NAD glycohydrolase 활성도 가지고 있다. Yac-2는 두 보존된 glutamic acids 사이인 221번 위치에 arginine를 소유하고 있다. 두 효소 활성에 대한 Arg-221의 중요성을 조사하기 위해 Arg-221이 Glutamic acid (R221E)와 Alanine (R221A)으로 돌연변이 되었다. 돌연변이체인 R221E와 R221A는 두 효소에 대해 야생형과 유사한 활성을 나타내었으며 이러한 결과는 Yac-2의 Arg-221이 ADP-ribosyltransferase와 NAD glycohydrolase의 활성에 필수적인 역할을 하지 않음을 시사해준다.

• 생명과학회지
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• 제15권2호
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• pp.304-310
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• 2005

생체세포에 대한 paraquat의 독작용은 superoxide dismutase 활성저해에 기인하는 것으로 알려져 있다. 그러나 세균이 paraquat에 매우 짧은 시간동안의 노출에 의하여서도 독작용을 받을 수 있는 것으로 검토됨에 따라 paraquat의 독작용의 하나로서 전자전달에 미치는 영향 중 NAD(H)의 산화 및 환원반응에 미치는 영향을 검토한 결과는 다음과 같다. 공시균의 원형질막 획분, rat mitocondria분산액 및 NAD-dependent dehydrogenase에 의한 산화 및 환원시 paraquat 첨가구에서 반응 Graph의 경사도가 더 컸으며, 반응 개시점 및 종결점이 대조구에 비해 낮은 결과로 반응이 가속화되는 결과를 볼 수 있었다. 반응을 경시적으로 NAD(H)의 함량변화로 검토한 결과에서도 원형질 막 획분과 rat mitocondria 분산액을 이용한 경우에 10분간의 NADH산화량이 대조구는 각각 960 mM, 1,187 mM이었으나 Paraquat 처리구는 각각 1,200 mM, 1,434 mM로 Paraquat 처리구가 반응 가속화 경향을 보였다. NAD(H) dependent dehydrogenase에 의한 NAD(H)의 산화 및 환원 반응에서도 대조구에 비하여 Paraquat 처리구가 초기반응의 가속화 및 총산화량의 증가를 보였다.

• Journal of Ginseng Research
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• 제24권1호
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• pp.35-40
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• 2000

홍삼에서 분리한 수용성 갈변물질의 항산화 활성을 linoleic acid, Ox-brain autoxidant, Fe$^{2+}$ ADP/NAD 및 cumene hydoperoxide 시스템에서 조사한 결과를 다음과 같다. 1. Linoleic acid :투석내액의 갈변물질보다 투석외액의 갈변물질 쪽에서 hydroperoxy radical 저해율이 높은 것으로 나타났으며, 이때 S-2의 산화저해율은 S-1, L시험구보다 적은 농도에서 저해율이 높았다. S-2의 산화저해율은 49,52, 62.44 및 97.5%로 나타나 첨가농도가 증가함에 따라 linoleic acid 산화저해율은 높았다. 2. Ox-brain autoxidant : 자동산화에의한 항산화활성을 조사한 결과, 투석내액의 갈변물질보다 투석외액의 갈변물질 쪽에서 자동산화에 의한 항산화활성이 높은 것으로 나타났다. 특히S-2의 항산화 활성은 22.5, 31.7, 31.9 및 33.5%로 나타났다. 3. Fe$^{2+}$ ADP/NAD 및 cumene hydroperoxide : 횐쥐 간의 microsome 분획을 분리하여 수용성 갈변물질의 ADP/NABP 및 cumene hydroperoxide 항산화 활성을 조사한 결과, 모든 시험구에서 반응 초기에 항산화 활성이 있으나, 반응 시간이 길수록 항산화 활성은 감소하였다. 그 활성의 순서는 S-2>S-1>L순으로 나타났으나 항산화 활성의 강도는 전체적으로 약하게 나타났다.