• Korean Journal of Microbiology
• /
• v.40 no.3
• /
• pp.221-225
• /
• 2004

Novel cytochrome P450 hydroxylase (CYP) genes were isolated and characterized from P. autotrophica cosmid DNA library using an actinomycete CYP-specific PCR product as a screening probe. The cosmids containing four unique CYP genes (pESK601, 602, 603, 604, 605) were identified, and the four CYP genes were completely sequenced to be homologous to other known Actinomycetes CYP genes involved in various secondary metabolic pathways. Among all novel actinomycete CYP genes found in P. autotrophica, the CYP genes present in pESK601 were revealed to be highly homologous to the CYP genes involved in polyene-type amphotericin and nystatin antibiotic biosynthesis. The nucleotide sequences of the CYP flanking region in pESK601 also revealed the polyene-type biosynthetic genes, implying the presence of a cryptic polyene-type antifungal biosynthetic gene cluster in P. autotrophica.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1995.10a
• /
• pp.153-155
• /
• 1995

To investigate the mechanism of the regulation of cytochrome P450IAl, the 5'-flanking region of a trout cytochrome P4501Al was cloned into the CAT basic expression vector at HindⅢ site. This trout Cytochrome P450IAl upstream DNA containing CAT construct was transfected into Hepa-1 cells .3MC treatment to hepa I cells transfected with trout P450IAl-CAT construct increased CAT protein and mRNA by 2.81 fold when it was compared with that of control. This increase CAT protein and mRNA was decreased by concomitantly treated flavonoids and aminopyrine. The level of CAT protein was 29.2-58.0% of 3MC stimulated CAT protein.

• Preventive Nutrition and Food Science
• /
• v.2 no.4
• /
• pp.328-332
• /
• 1997

The ripe fruit of Hass avocado contains one of the highest elvels of cytochrome P-450 protein found in the plant kingdom. To determine whether wounded roots of avocado contain P-450 protein, the roots of avocado were wounded by slicing, and then allowed to incubate in sealed plastic bags, in 0.4M mannitol, and in the solution to make protoplast preparation containing cellulysin and macerase during the specified times. The microsomal proteins were extracted from the samples, separated by SDS-PAGE, and then subjected to Western blot analysis using polyclonal antibodies which are generated against the CYP71A1 protein. wounded roots in sealed bags produced CYP71A1 within 6 hours after cutting, and those in 0.4M mannitol did not produce CYP71A1 even after 72 hours, but those in the protoplast preparation by cellulysin and macerase induced and produced CYP71A was induced in only 24 hours. These results indicate that CYP 71A1 plays a role for wound healing for root tissue o avocado, and would-inducible P-450 protein was not detected in the mannitol solution by preventing a synthesis of ethylene in a liquid state, but the softening of tissues by cellulysin and macerase to make protoplast preparation was involved in an activation of CYP 71A1 even in the liquid state.

• Journal of Preventive Medicine and Public Health
• /
• v.32 no.1
• /
• pp.88-94
• /
• 1999

Objectives. In order to gain a better understanding of the mechanism of DMF toxicity, recent studies have focused on hepatic drug metabolizing enzymes. In this study, we investigated the effects of DMF on the induction of P450 and the activities of other related enzymes in rat liver microsomes. Methods. DMF was administered to male Sprague Daweley rats by intraperitoneal injection at 0(control), 450(D1), 900(D2), 1,800(D3) mg DMF/kg body weight in olive oil once a day for three days. Hepatic P450 was measured by method of Omura and Sato. We evaluated selective assays for the three drug metabolizing cytochrome P450 isoenzymes 1A1, 2B1 and 2E1. Results. The content of microsomal protein, P450 and b5 were tended to be decreased in DMF treated group, but they were not statistically significant. The activity of NADPH-cytochrome P450 reductase was significantly increased dose dependently(p<0.01), but the activity of NADH-b5 reductase was decreased in the treated group(p<0.01). The activities of PROD and EROD were not significant between control and treated group. The activities of pNPH in the DMF treated groups were higher than that of the control group(p<0.01). When Western immunoblottings were carried out utilizing three monoclonal antibodies which were specific against P4501A1/1, P4502B1/2 and P4502E1, the strong density band corresponding to P4502E1 was observed with the microsomes obtained from the rats treated with DMF. But there were no significant increased in the P4501A1/2 and P4502B1/2 band densities in immunoblotting. Conclusions. These result suggested that P4502E1 was inducible by DMF and P4502E1 isozyme might be responsible for the hydroxylation of DMF to HMMF.

• Journal of Sasang Constitutional Medicine
• /
• v.24 no.4
• /
• pp.84-91
• /
• 2012

Objectives : The purpose of this study is to investigate the inhibitory or inductive potentials of Yuldahanso-tang (YDT) and Chungsimyonja-tang (CST), herbal formulas for Taeeumin, on cytochrome P450 (CYP450) drug metabolizing enzyme. The mechanisms for the herbal formula-drug interaction has not been well reported in spite of the chance for co-administration with conventional drugs. Methods : To evaluate the interaction potential of YDT-drug or CST-drug, the fluorescence-based enzyme assays on CYP450 isozymes including CYP3A4, CYP2C19, CYP2D6 and CYP2E1 were established in vitro. The inhibitory effects of herbal formulas were characterized with $IC_{50}$ values. Results : YDT showed inhibitory effects on CYP2D6 and CYP2E1-mediated metabolism, while it exhibited week inhibition on CYP3A4 and CYP2C19 relatively. CST exerted relatively weak inhibitory effects on the four CYP450 isozymes compared to that of YDT. Conclusions : These results suggest that the herbal formula-drug interaction could be occur when YDT are co-administered with drugs mediated by CYP2D6 or CYP2E1.

• Food Science and Biotechnology
• /
• v.14 no.2
• /
• pp.297-300
• /
• 2005

Effects of allyl sulfides on kinetic behavior of cytochrome P450 1 (CYP1)-catalyzed ethoxyresorufin O-deethylase (EROD) activity were studied using microsomes from benzo[a]pyrene-treated human hepatoma cells. Apparent $K_m$ and $V_{max}$ values were calculated as $2.8\;{\mu}M$ and $3.0\;{\mu}mol$ resorufin/min/mg protein based on Lineweaver-Burk plot of microsomal EROD activity, respectively. Diallyl disulfide (DADS) and diallyl trisulfide (DATS) affected $K_m$ and $V_{max}$ values of EROD activity and acted as mixed-type inhibitors for CYP1 isozymes. Apparent Ki values of DADS and DATS were calculated as 1.07 and 0.88 mM, respectively, by re-plotting slopes of Lineweaver-Burk plot and inhibitor concentrations.

• Proceedings of the Korean Society of Toxicology Conference
• /
• 2003.05a
• /
• pp.77-77
• /
• 2003

We have previously shown that 2, 3' , 4, 5' -tetramethoxystilbene(TMS), a trans-stilbene analogue, is one of the most potently selective inhibitor of recombinant human cytochrome P450 1B1 in vitro. In the present studies, the effects of TMS on the expression of cytochrome P450 1B1 were investigated in human mammary cell lines such as MCF-7 and MCF-10A. (omitted)

• Archives of Pharmacal Research
• /
• v.21 no.1
• /
• pp.35-40
• /
• 1998

Human cytochrome P450 4F2 shows high regioselectivity in hydroxylation of stearic acid and leukotriene $ B_4.$ As a first step of its regulation study, human cytochrome P450 4F2 genomic DNA was isolated from liver of a person who was administered clofibrate for 10 years. From Southern hybridization, restriction enzyme digestion and sequencing experiments, isolated genomic DNA fragment was found to contain around 32 Kb DNA and more than 20 Kb of $5^I$ end regulatory region. Sequences of the structural gene region revealed exon 1 and exon 2. Further regulation studies would elucidate the feedback mechanisms of the oxidative degradation of fatty acids, inflammatory response and the clearance of leukotriene B4 in the liver. Furthermore, regulation study of this gene could explain the species difference in responses to peroxisome proliferator and help in the safety evaluation of peroxisome proliferating chemicals to human being.

• Biomolecules & Therapeutics
• /
• v.32 no.4
• /
• pp.474-480
• /
• 2024

Streptomyces avermitilis genome includes 33 genes encoding monooxygenation-catalyzing cytochrome P450 enzymes. We investigated the structure of CYP107P2 and its interactions with terpenoid compounds. The recombinant CYP107P2 protein was expressed in Escherichia coli and the purified enzyme exhibited a typical P450 spectrum upon CO-binding in its reduced state. Type-I substrate-binding spectral titrations were observed with various terpenoid compounds, including α-pinene, β-pinene, α-terpinyl acetate, and (+)-3-carene. The calculated binding affinities (Kd) ranged from 15.9 to 50.8 µM. The X-ray crystal structure of CYP107P2 was determined at 1.99 Å resolution, with a well-conserved overall P450 folding conformation. The terpenoid compound docking models illustrated that the structural interaction between monoterpenes and CYP107P2, with the distance between heme and terpenes ranging from 3.4 to 5.4 Å, indicates potential substrate binding for P450 enzyme. This study suggests that CYP107P2 is a Streptomyces P450 enzyme capable of catalyzing terpenes as substrates, signifying noteworthy advancements in comprehending a novel P450 enzyme's involvement in terpene reactions.

• Korean Journal of Veterinary Research
• /
• v.38 no.1
• /
• pp.17-28
• /
• 1998

Drug-metabolizing activities of Korean native cattle and swine were investigated from viewpoints of the cytochrome P-450's level, their dependent mixed function oxidase activities, the reactive oxygen species formation and cytosolic enzyme acitivities from each liver homogenates. Level of cytochrome P-450 in the liver microsome of Korean native cattle was $0.28{\pm}0.05nmole/mg$ and that in pigs $0.35{\pm}0.03nmole/mg$. Level of cytochrome $b_5$ of Korean native cattle was $0.24{\pm}0.06nmole/mg$, and that of pigs $0.2{\pm}0.05nmole/mg$, showing no difference between two species. NADPH P-450 reductase were higher in Korean native cattle ($58.3{\pm}5.3nmole/mg/min$) than in pigs ($29.9{\pm}3.8nmole/mg/min$)(p<0.01). The activities of cytochrome P-450 dependent monooxygenases such as ethoxyresorufin O-deethylase (cattle, $96.5{\pm}12.5nmole/mg/min$ ; pigs, $13.6{\pm}2.1nmole/mg/min$), N-benzphetamine N-demethylase (cattle, $5.23{\pm}0.82nmole/mg/min$ ; pigs, $0.76{\pm}0.3nmole/mg/min$) and aniline hydroxylase (cattle, $0.95{\pm}0.1nmole/mg/min$ ; pigs, $0.33{\pm}0.08nmole/mg/min$) were much higher in Korean native cattle than in swine(p<0.01). However, the activity of testosterone $7{\alpha}$-hydroxylase was higher in swine ($90.4{\pm}1.2nmole/mg/min$) than cattle (cattle, $32.8{\pm}1.2nmole/mg/min$). Interestingly, testosterone $16{\alpha}$-hydroxylase, a marker enzyme for P-450 IIA was not detected in both animal species. These results suggest that Korean native cattle and pigs have high contents of P-450 IA1 and P-450 IIIA. Total sulfhydryl compound (cattle, $10.3{\pm}1.1nmole/mg$ ; Pigs, $14.5{\pm}1.8nmole/mg$) and glutathione related enzymes except glutathione reductase (cattle, $38.1{\pm}7.9nmole/mg/min$; swine, $22{\pm}3.6nmole/mg/min$) showed higher levels in swine than in Korean native cattle. Superoxide dismutase (cattle, $7.64{\pm}0.84nmole/mg/min$ ; pigs, $4.47{\pm}0.94nmole/mg/min$) and catalase (cattle, $30.4{\pm}3.7nmole/mg/min$ ; pigs, $17.2{\pm}1.8nmole/mg/min$) were remarkably higher in Korean native cattle than in swine (p<0.05).