• Toxicological Research
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• v.18 no.3
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• pp.249-257
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• 2002

Cytochrome P450 3As such as 3A4 and 3A5 metabolize a wide range of pharmaceutical compounds. The vectors for the expression of fusion protein containing an N-terminal human P450 3A4 or P450 3A5 sequences and a C-terminal rat NADPH-cytochrome P450 reductase moiety were constructed. These plasmids were used to express the fusion protein in Escherichia coli DH5$\alpha$ cells. High levels of expression were achieved (100~200 nmol/liter) and the expressed fusion protein in E. coli membranes were catalytically active for nifedipine oxidation, a typical enzymatic activity of P450 3A4. The NADPH-P450 reductase activities of these fusion protein were also determined by measuring reduction of cytochrome c. To fine a specific Inhibitor of P450 3A4 from naturally occurring chemicals, a series of isothiocyanate compounds were evaluated for the inhibitory activity of P450 using the fusion proteins in E. coli membranes. Of the five isothiocyanates (phenethyl isothiocyanate, phenyl isothiocyanate, benzol isothiocyanate, benzoyl isothiocyanate and cyclohexyl isothiocyanate) tested, benzoyl isothiocyanate showed a strong inhibition of P450 3A4 with an $IC_{50}$value of 2.8 $\mu\textrm{M}$. Our results indicate that the self-sufficient fusion protein will be very useful tool to study the drug metabolism and benzyl isothiocyanate may be valuable for characterizing the enzymatic properties of P450 3A4.

• Biomedical Science Letters
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• v.7 no.2
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• pp.65-70
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• 2001

We have carried out PCR reactions to investigate if cytochrome P450 (P450) enzymes (1A1, 1A2, 2C8, 2E1 and 3A4), which are well hewn to be the key enzymes in detoxification process and/or synthesis of steroids in the liver, are expressed in the human brain, too. P450 1A1, 2C8 and 2E1 were expressed clearly. However, P450 1A2 and 3A4 were not detectable. Their expression levels in the human brain could be extremely low or they were not expressed at all. One base substitution at nucleotide 290 (A->G) was identified in P450 1A1. It is suspected to be an individual polymorphism. Our results should contribute to the better understanding of the role of cytochrome P450 enzymes in the human brain.

• The Journal of Internal Korean Medicine
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• v.34 no.4
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• pp.375-383
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• 2013

Objectives : This study was performed to investigate the metabolic site of some medicinal herbs in the liver associated with CYP (Cytochrome P450). Methods : Cytochrome P450 is the major enzymes involved in drug metabolism and bioactivation. CYP3A4 and CYP2D6, the major CYP isoforms in humans, catalyse the major proportion of drugs available on the market. Scintillation proximity assay (SPA) is often used in studies to identify compounds that inhibit CYP3A4 and CYP2D6. 28 herbal extracts and radioisotopes were attached competitively to SPA beads, and followed by measuring the remaining radioisotopes in the medium. Erythromycin and dexamethasone, inhibitors of CYP3A4 and CYP2D6, were used as controls respectively. Results : Most of the 28 herbal extracts showed dose-dependent affinity to the CYP3A4 while some of the herbs showed affinity to the CYP2D6. Conclusions : These results suggest that most of the 28 herbal extracts are metabolized safely in the liver, combined with CYP3A4 and CYP2D6.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.149.3-150
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• 2003

CYP3A4 is the most abundant human CYP and oxidizes a diversity of substrates. including various drugs. steroids. and carcinogens. A variety of metal ions are known to affect microsomal monooxygenase activities. Effects of a series of divalent metal ions on the CYP3A4-catalyzed reaction of reconstituted system containing purified CYP3A4. NADPH-P450 reductase (NPR), and cytochrome b5 (b5) were examined. (omitted)

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.53-53
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• 2002

Inhibitory effects of Cu$\^$2＋/ on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn$\^$2＋/, Mg$\^$2＋/, Mn$\^$2＋/, Ca$\^$2＋/, and Co$\^$2＋/ had no apparent effects on the activities of microsomal P450s. Cu$\^$2＋/ inhibited the reactions catalyzed by purified P450s lA2 and 3A4 with IC$\sub$50/ values of 5.7 and 8.4 ${\mu}$M, respectively.(omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.100-100
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• 1993

Antiestrogen은 에스트로젠 의존성 유방암 치료에 사용되는 약물로써 표적 세포 내에서 에스트로젠 수용체와 작용하여 세포 증식 억제 작용을 나타내고 동시에 에스트로젠 수용체와는 구분되는 소포체 분획의 antiestrogen specific binding site (AEBS) 와도 결합을 하는 것으로 알려져 있다. 그러나, 아직 이 AEBS의 생리적 또는 약리적 기능은 밝혀져 있지 않다. 따라서 본 실험에서는 AEBS의 기능을 조사하기 위하여 cytochrome P45O III 효소군과 AEBS와의 관계를 자옹 백서를 이용하여 면역 화학 반응 실험 및 경쟁적 결합 반응 실험을 하였고, 그 결과는 다음과 같다. 1) AEBS에 대해서 SKF-525A와 metyrapone은 결합 능력을 나타내었다. 2) 자성쥐에서는 주령이 증가함에 따라 cytochrome P450양이 감소하였다. 3) 자옹성쥐 모두에서 phenobarbital 처치에 의해 cytochrome P450 III 효소양이 증가하였고, AEBS도 증가하였다. 4) 웅성쥐에서는 testosterone에 의하여 AEBS가 증가하였다. 5) 자웅성쥐 모두에 tamoxifen 관류시 cytochrome P450 III 효소양이 증가하였고 estradiol과 병용 관류시에는 tamonifen 단독 관류시보다 감소하였다. 이상의 결과에서 tamoxifen이 cytochrome P450 III을 유도할 수 있는 것으로 사료되며 cytochrome P450 III 효소군과 안티에스트로젠 결합부위와 밀접한 관련이 있는 것으로 생각된다.

• Toxicological Research
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• v.12 no.2
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• Toxicological Research
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• v.11 no.2
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• pp.205-213
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• 1995

This study was undertaken to investigate the effects of organic solvents on xenobiotic metabollzing enzyme system in vivo by meaas of experimental conditions i.e. (1) single group which was treated by benzene (B), toluene (T) and xylene (X), respectively, (2) combination group which was treated by mixture of benzene+toluene (BT), benzene+xylene (BX), and toluene+xylene (TX), respectively, (3) mixture group which was treated by benzene+ toluene+xylene mixture (M), and to interpreat the interaction between the organic solvents metabolizing enzymes. 1. The contents of cytochrome P-450 in liver microsomes were increased (p < 0.01) in organic solvents treated groups, and the contents of cytochrome P-450 were increased by following order of B < T < M < BT=BX < X < TX. 2. The activity of cytochrome P-450 dependent AHHase was significantly higher in organic solvents treated groups than in control group (p < 0.01), and the activity of AHHase was increased by following order of B < T < BT=BX=TX=xylene < M. 3. The activity of NADPH P-450 reductase was significantly higher in organic solvents treated groups than in control group (p < 0.01), and the order of M < combinated group < X < T

• Archives of Pharmacal Research
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• v.18 no.3
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• pp.179-182
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• 1995

Previous study showed that $CCl_4$ administration evoked a rapid decrease in cytochrome $P_{450}$ 2E1 protein soon after the exposure due to posttranslational inhibition(Biochem. Biophys. Res. Commun. 179:449-454, 1991). In this report, aniline hydroxylase and the amounts of immunoreactive $P_{450}$ 2E1 were rapidly decreased during day 1 to 2 and recovered during day 3 to 4 after a single dose of $CCl_4$. The activity of pentoxyresorufin-O-dealkylase was also suppressed at day 1 and began to repair from day 2. However, the decrease in immunoreactive $P_{450}$ 2C content was not observed. The decreases in $P_{450}$ 2E1 enzyme activity and immunoreactive protein by acute $CCl_4$ treatment were accompanied by a decline in $P_{450}$ 2E1 mRNA level. The data thus suggested a pretranslational reduction of $P_{450}$ 2E1 during day 1 to 2 after acute $CCl_4$ treatment.

• Toxicological Research
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• v.11 no.2
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• pp.329-335
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• 1995

Incubation of aflatoxin $B_1$ $(AFB_1)$ with microsomes isolated from human liver number 110 yielded two metabolite peaks which were aflatoxin $Q_1$ $(AFQ_1)$ and $(AFB_1)$-exo-8, 9-epoxide (exo-epoxide) in high performance liquid chromatography. Production ratio of $AFQ_1$ to exo-epoxide was 2.43$\pm $0.04. Metabolism of $(AFB_1)$ to $(AFQ_1)$ and exo-epoxide was inhibited by troleandomycin in a same degree although troleandomycin was not activated as a mechanism-based inhibitor. The inhibitory effect was dependent upon either the incubation time with $(AFB_1)$ or the preincubation time before the addition of $(AFB_1)$. Incubation of troleandomycin and NADPH by the microsomes resulted in the formation of a cytochrome P 450 (P450)-metabollc intermediate (MI) complex and the level was approximately 80% of total P450 3A4 in the microsomes. This figure was similar to that of the inhibitory effect of troleandomycin on $AFB_1$ metabolism. Glutathione which was reported that it prevented the formation of MI complex in rat liver microsomes did not inhibit the formation of MI complex in human liver microsomes. These results suggested that the inhibitory effect of troleandomycin on $AFB_1$ metabolism is due to the formation of MI complex with P450 3A4. And the reaction mechanism of troleandomycin by human liver microsomes might be dlfferent from that one by rat liver microsomes.