(1/68) Purified fusion enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 oxidoreductase.

A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.  (+info)

(2/68) Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts.

Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N. The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22. Enzymatic assays were carried out in vitro with the isolated microsomes. P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron. P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays. Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23. 8 min(-1) for the herbicide chlortoluron, respectively. Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously. The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed.  (+info)

(3/68) Inhibitory effects of 1,4-naphthoquinone derivatives on rat cytochrome P4501A1-dependent monooxygenase activity in recombinant yeast microsomes.

We reported previously that various naphthoquinone derivatives inhibited cytochrome P450-dependent monooxygenase of liver and placenta microsomes [Muto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 487-494]. To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P450 species. In the present study, the inhibitory effects of six derivatives of 1,4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P4501A1. Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5,8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition. Judging from the inhibitor constant (K(i)), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P4501A1 is in the order: 2-CH(3)-5-OH-NQ > 2-CH(3)-NQ > NQ >> 2-OH-NQ. On binding with P4501A1, 2-CH(3)-5-OH-NQ, 2-CH(3)-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively. These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity.  (+info)

(4/68) Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes.

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.  (+info)

(5/68) Maturational changes in CYP2D16 expression and xenobiotic metabolism in adrenal glands from male and female guinea pigs.

CYP2D16 is expressed at high levels in the zona reticularis (ZR) of guinea pig adrenal glands and contributes to adrenal metabolism of xenobiotics. Studies were done to evaluate the effects of age and gender on adrenal CYP2D16 expression and xenobiotic metabolism. In both male and female guinea pigs at 1, 7, 14, or 30 weeks of age, in situ hybridization and immunohistochemistry confirmed that CYP2D16 was highly localized to the ZR of the adrenal gland. The steroidogenic P450 isozyme, CYP17, by contrast, was expressed in both the zona fasciculata and ZR. The intensity of CYP2D16 staining was not age- or gender-dependent. However, the proportion of each adrenal gland comprised by ZR and thus expressing CYP2D16 increased with aging in both sexes and was greater in males than in females. The rates of metabolism of bufuralol, a CYP2D-selective substrate, by adrenal microsomal preparations generally correlated with the amount of ZR (and CYP2D16) in the gland. Thus, adrenal xenobiotic-metabolizing activities were greater in males than in females at all ages and increased with aging in males. However, the rates of bufuralol metabolism declined in sexually mature females (14 weeks) from the levels found in prepubertal females (7 weeks) and then increased markedly in retired breeders (30 weeks), suggesting an inhibitory effect of estrogens on enzyme activity. The results indicate that the age and gender differences in adrenal CYP2D16 content are largely determined by differences in the size of the ZR rather than the concentrations of CYP2D16 within cells of the ZR. However, adrenal xenobiotic-metabolizing activities in females seem to be further modulated by an inhibitory effect of estrogens.  (+info)

(6/68) P450 enzyme expression patterns in the NCI human tumor cell line panel.

Cytochrome P450 (P450) enzyme expression patterns were determined for a panel of 60 human tumor cell lines, representing nine tumor tissue types, used by the National Cancer Institute (NCI) Anticancer Drug Screening Program. All 60 tumor cell lines displayed significant P450 activity, as well as P450 reductase activity, as determined using the general P450 substrate 7-benzyloxyresorufin. Cell line-specific P450 enzyme patterns were observed using three other P450 substrates, 7-ethoxycoumarin, coumarin, and 7-ethoxyresorufin, each of which was metabolized at a low rate. Using a pattern-matching computer program, COMPARE, correlative relationships were investigated between the arrays of P450 activities and the patterns of cytotoxicity exhibited by a large group of anticancer agents of proven or potential clinical utility. Significant negative correlations between the patterns of P450-dependent 7-benzyloxyresorufin metabolism activity and cell line chemosensitivity were observed for 10 standard anticancer agents (including 6 alkylating agents) and 55 investigational compounds, suggesting a role for P450 metabolism in the inactivation of these agents. Negative correlations between 7-ethoxycoumarin O-deethylation and cell line chemosensitivity to a group of topoisomerase inhibitors were also seen, again suggesting P450-dependent drug inactivation. P450 enzyme profiling may thus aid in interpreting the patterns of drug sensitivity and resistance in the NCI tumor cell panel, and may facilitate the identification of anticancer agents whose activity can be altered via cytochrome P450 metabolism.  (+info)

(7/68) Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.  (+info)

(8/68) Engineering herbicide metabolism in tobacco and Arabidopsis with CYP76B1, a cytochrome P450 enzyme from Jerusalem artichoke.

The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites.  (+info)

*  List of MeSH codes (D08)
... map kinase kinase 7 MeSH D08.811.913.696.620.682.725.300 --- proto-oncogene proteins c-fes MeSH D08.811.913.696.620.682.725.400 ... map kinase kinase 7 MeSH D08.811.913.696.620.682.700.567 --- mitogen-activated protein kinases MeSH D08.811.913.696.620.682. ... cholesterol 7 alpha-hydroxylase MeSH D08.811.682.690.708.170.915.212 --- cholesterol side-chain cleavage enzyme MeSH D08.811. ... cholesterol 7 alpha-hydroxylase MeSH D08.244.453.915.212 --- cholesterol side-chain cleavage enzyme MeSH D08.244.453.915.400 ...
List of MeSH codes (D08) - Wikipedia  List of MeSH codes (D08) - Wikipedia
... map kinase kinase 7 MeSH D08.811.913.696.620.682.725.300 --- proto-oncogene proteins c-fes MeSH D08.811.913.696.620.682.725.400 ... map kinase kinase 7 MeSH D08.811.913.696.620.682.700.567 --- mitogen-activated protein kinases MeSH D08.811.913.696.620.682. ... cholesterol 7 alpha-hydroxylase MeSH D08.811.682.690.708.170.915.212 --- cholesterol side-chain cleavage enzyme MeSH D08.811. ... cholesterol 7 alpha-hydroxylase MeSH D08.244.453.915.212 --- cholesterol side-chain cleavage enzyme MeSH D08.244.453.915.400 ...
more infohttps://en.wikipedia.org/wiki/List_of_MeSH_codes_(D08)
NAVER Academic > Search...  NAVER Academic > Search...
Reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells. II. Enhancement of the alkylating activity of AZQ ... 7-Alkoxycoumarin O-Dealkylase, antagonists & inhibitors, Aminopyrine N-Demethylase, Animals, Cytochrome P-450 Enzyme System, ...
more infohttps://academic.naver.com/search.naver?field=3&query=BIOCHEMICAL+PHARMACOLOGY+44%EA%B6%8C+8%ED%98%B8
Enzyme-kinetic and immunochemical characteristics of mouse cDNA-expressed, microsomal, and purified CYP1A1 and CYP1A2. -...  Enzyme-kinetic and immunochemical characteristics of mouse cDNA-expressed, microsomal, and purified CYP1A1 and CYP1A2. -...
Western blot/densitometry analysis revealed a 4.8 times lower binding of Mab 1-7-1 to cDNA-expressed CYP1A2 than to CYP1A1. The ... and 7-ethoxyresorufin o-deethylase (EROD) were estimated in microsomes of Hep G2 cells infected with a recombinant vaccinia ... 1-7-1 displayed a striking difference between its immunoblotting and inhibitory effects. ... Kinetics of benzo[alpha]pyrene hydroxylase (AHH), 7-methoxyresorufin o-demethylase (MROD), and 7-ethoxyresorufin o-deethylase ( ...
more infohttps://www.semanticscholar.org/paper/Enzyme-kinetic-and-immunochemical-characteristics-Tsyrlov-Goldfarb/b081e40dbfb8ede74f791450da4cbeb617720f61
Effect of the metabolic capacity in rat liver S9 on the positive results of <i>in vitro</i>...  Effect of the metabolic capacity in rat liver S9 on the positive results of <i>in vitro</i>...
Increases in P450 content and alkoxycoumarin dealkylase activity in PB-BNF-induced rat S9. a, P450 contents; b, alkoxycoumarin ... Consequently, alkoxycoumarin O-dealkylase activity known as markers for CYP activity and protein expression of CYP1A1, CYP1A2, ... Based on the results of the cell growth assay and in vitro micronucleus test of the dose range-finding test, 3 to 7 dose levels ... O-dealkylase (ACD) activities; Open bars, untreated rat S9; Gray bars; combination of phenobarbital and 5,6-benzoflavone- ...
more infohttps://www.jstage.jst.go.jp/article/jts/44/3/44_145/_html/-char/en
Mch - acetyl-beta-methylcholine | AcronymAttic  Mch - acetyl-beta-methylcholine | AcronymAttic
O-Dealkylase 7-Chloro-4-nitrobenzofurazan 7-Chloro-7-deoxylincomycin 7-Ethoxycoumarin Dealkylase 7-Ethoxycoumarin Hydroxylase 7 ... Eine benzopyrene-Ableitung mit krebserregender und mutagener Aktivit?t. 7,8-BaP-9,10-Diol Epoxide 7,8- ... Monooxygenase 7-Ethoxycoumarin O-De-Ethylase 7-Ethoxycoumarin O-Deethylase 7-Hydroxycoumarin UDP Glucuronyltransferase 7- ... Hydroxycoumarins 7-O-Methylnogarol 7-OMEN 7-Phosphat-Synthetase, 3-Deoxyarabinoheptulosonate 70143, Bayer 72 kD Gelatinase 740 ...
more infohttps://www.acronymattic.com/acetyl_beta_methylcholine-
Cytochrome P-450 activity in hepatocytes following cryopreservation and monolayer culture.  - PubMed - NCBI  Cytochrome P-450 activity in hepatocytes following cryopreservation and monolayer culture. - PubMed - NCBI
PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.
more infohttps://www.ncbi.nlm.nih.gov/pubmed/3876098?dopt=Abstract
Activities of glutathione-S-transferase and ethoxycoumarin-O-deethylase in tissues of camels, sheep, goats and rats<...  Activities of glutathione-S-transferase and ethoxycoumarin-O-deethylase in tissues of camels, sheep, goats and rats<...
TY - JOUR. T1 - Activities of glutathione-S-transferase and ethoxycoumarin-O-deethylase in tissues of camels, sheep, goats and rats. AU - El Sheikh, H. A.. AU - Ali, B. H.. AU - Homeida, A. M.. AU - Hapke, H. J.. PY - 1991. Y1 - 1991. N2 - 1. The activities of the drug metabolizing enzymes ethoxycoumarin-O-deethylase, glutathione-S-transferase, and protein concentrations were measured in vitro in the liver, kidney and duodenal mucosa of camels, sheep, goats and rats. 2. Enzyme activities were generally higher in the liver than in the kidney and duodenal mucosa in the four species studied. 3. The activities of ethoxycoumarin-O-deethylase and glutathione-S-transferase in liver of male kids were about one third and half of that in adult male goats, respectively.In the kidney and duodenal mucosa of male kids, the activity of glutathione-S-transferase was about 70% and 53% of that in the mature male goat, respectively. In the latter tissues, however, there was no detectable activity of ...
more infohttps://squ.pure.elsevier.com/en/publications/activities-of-glutathione-s-transferase-and-ethoxycoumarin-o-deet
Monoclonal antibody-directed determination of cytochrome P-450 types expressed in a human lymphoblastoid cell line  Monoclonal antibody-directed determination of cytochrome P-450 types expressed in a human lymphoblastoid cell line
Partial inhibition by the MAb 1-7-1, however, indicates that at least two forms of cytochrome P-450 catalyze 7-ethoxycoumarin O ... A sensitive MAb 1-7-1-based radioimmunoassay also directly demonstrates the presence in these cells of a MAb 1-7-1-specific ... The monoclonal antibody inhibition determined that a single MAb 1-7-1-sensitive type of cytochrome P-450 is responsible for all ... Cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities of a cloned line of ...
more infohttps://vivo.scripps.edu/display/endnote67315
Xenobiotic-metabolizing enzyme activity in human non-small-cell derived lung cancer cell lines<...  Xenobiotic-metabolizing enzyme activity in human non-small-cell derived lung cancer cell lines<...
TY - JOUR. T1 - Xenobiotic-metabolizing enzyme activity in human non-small-cell derived lung cancer cell lines. AU - Falzon, Miriam. AU - McMahin, James B.. AU - Schuller, Hildegard M.. PY - 1986/2/15. Y1 - 1986/2/15. N2 - Human lung cancer cell lines in culture were investigated for the expression of monooxygenase and other xenobiotic-metabolizing enzyme activities. Two brochiolo-alveolar carcinoma derived cell lines (NCI-H322 and NCI-H358) and two small-cell carcinoma derived cell lines (NCI-H128 and NCI-H69) were used. Previous work has shown that NCI-H322 has ultrastructural features of Clara cells while NCI-H358 shows characteristics of alveolar type II cells [Schuller et al., Proc. Am. Ass. Cancer Res. 26, 27 (1985)]. NCI-H128 and NCI-H69 show very poor differentiation of cytoplasmic organelles. Cytochrome P-450 levels were spectroscopically detectable only in NCI-H322. Both NCI-H322 and NCI-H358, but not NCI-H69 and NCI-H128, exhibited aryl hydrocarbon hydroxylase (using benzo[a] pyrene ...
more infohttps://researchexperts.utmb.edu/en/publications/xenobiotic-metabolizing-enzyme-activity-in-human-non-small-cell-d
ONKI3 | Medical Subject Headings - MeSH  ONKI3 | Medical Subject Headings - MeSH
7-Alkoxycoumarin O-Dealkylase, 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide... ... 6-Aminocaproic Acid, 6-Aminonicotinamide, 6-Cyano-7-nitroquinoxaline-2,3-dione, 6-Ketoprostaglandin F1 alpha, 6-Mercaptopurine ... 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific), 3-Deazauridine, 3-Deoxy-7-Phosphoheptulonate Synthase, 3-Hydroxyacyl CoA ... 5-Tetrahydro-8-chloro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol... ...
more infohttp://onki.fi/en/browser/overview/mesh?c=http://www.yso.fi/onto/mesh/D008271&l=en
Diurnal difference in CAR mRNA expression | Nuclear Receptor | Full Text  Diurnal difference in CAR mRNA expression | Nuclear Receptor | Full Text
Furukawa T, Manabe S, Ohashi Y, Sharyo S, Kimura K, Mori Y: Daily fluctuation of 7-alkoxycoumarin O-dealkylase activities in ... showed that hepatic P450-dependent monooxygenase activities measured by the O-dealkylation of 7-alkoxycoumarin fluctuate daily ... Noshiro M, Nishimoto M, Okuda K: Rat liver cholesterol 7α-hydroxylase. Pretranslational regulation for circadian rhythm. J Biol ... and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP. Mol ...
more infohttps://nuclear-receptor.biomedcentral.com/articles/10.1186/1478-1336-2-6
  • Cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities of a cloned line of human lymphoblastoid AHH-1 cells are inhibited by a monoclonal antibody (MAb 1-7-1) prepared to a 3-methylcholanthrene-induced rat liver cytochrome P-450. (scripps.edu)