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Tsutomu Shimada - One of the best experts on this subject based on the ideXlab platform.
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Roles of NADPH-P450 reductase in the O-deethylation of 7-Ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli.
Protein Expression and Purification, 2000Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, F. Peter Guengerich, Kiyoshi InoueAbstract:Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-Ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-Ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-Ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was
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roles of nadph p450 reductase in the o deethylation of 7 ethoxycoumarin by recombinant human cytochrome p450 1b1 variants in escherichia coli
Protein Expression and Purification, 2000Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi InoueAbstract:Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-Ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-Ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-Ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes, (C) 2000 Academic Press.
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roles of nadph p450 reductase in the o deethylation of 7 ethoxycoumarin by recombinant human cytochrome p450 1b1 variants in escherichia coli
Protein Expression and Purification, 2000Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi InoueAbstract:Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-Ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-Ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-Ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.
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prediction of human liver microsomal oxidations of 7 ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome p 450 enzymes
Drug Metabolism and Disposition, 1999Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi YamazakiAbstract:Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-Ethoxycoumarin and chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities ( V max / K m ratio) in catalyzing 7-Ethoxycoumarin O -deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-Ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles ( V max / K m ratio) for 7-Ethoxycoumarin O -deethylation and chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.
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highly sensitive high performance liquid chromatographic assay for coumarin 7 hydroxylation and 7 ethoxycoumarin o deethylation by human liver cytochrome p450 enzymes
Journal of Chromatography B: Biomedical Sciences and Applications, 1999Co-Authors: Hiroshi Yamazaki, Masaaki Tanaka, Tsutomu ShimadaAbstract:A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-Ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-Ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-Ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.
Hiroshi Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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prediction of human liver microsomal oxidations of 7 ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome p 450 enzymes
Drug Metabolism and Disposition, 1999Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi YamazakiAbstract:Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-Ethoxycoumarin and chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities ( V max / K m ratio) in catalyzing 7-Ethoxycoumarin O -deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-Ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles ( V max / K m ratio) for 7-Ethoxycoumarin O -deethylation and chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.
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highly sensitive high performance liquid chromatographic assay for coumarin 7 hydroxylation and 7 ethoxycoumarin o deethylation by human liver cytochrome p450 enzymes
Journal of Chromatography B: Biomedical Sciences and Applications, 1999Co-Authors: Hiroshi Yamazaki, Masaaki Tanaka, Tsutomu ShimadaAbstract:A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-Ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-Ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-Ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.
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Prediction of human liver microsomal oxidations of 7-Ethoxycoumarin and chlorzoxazone using kinetic parameters of recombinant cytochrome P450 enzymes. Drug Metab Dispos 27:1274–1280.
1999Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi YamazakiAbstract:This paper is available online at http://www.dmd.org ABSTRACT: Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-Ethoxycoumarin and chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities (V max /K m ratio) in catalyzing 7-Ethoxycoumarin O-deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-Ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles (V max /K m ratio) for 7-Ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples. Multiple forms of cytochrome P-450 (CYP) 2 exist in liver microsomes and these CYP forms play important roles in the oxidation of structurally diverse xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens as well as endobiotic chemicals, including steroids, fatty acids, fat-soluble vitamins, and prostaglandins Several studies have reported that two or more CYP enzymes are able to oxidize xenobiotic and endobiotic chemicals at the same position of the molecules with different affinities (i.e., V max /K m ratio) in human liver microsomes 1274 predicted with kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and 3A4 enzymes Materials and Methods Chemicals. 7-Ethoxycoumarin was obtained from Aldrich Chemical Co. (Milwaukee, WI) and 7-hydroxycoumarin from Katayama Chemical Co. (Osaka, Japan). Chlorzoxazone and its 6-hydroxylated metabolite were donated by Dr. F. P. Guengerich of Vanderbilt University. Other reagents and chemicals used in this study were obtained from sources as described previously or were of highest qualities commercially available Enzyme Preparation. Human liver samples were obtained from organ donors or patients undergoing liver resection as described previously Recombinant CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11 expressed in microsomes of T. ni cells infected with a baculovirus containing human CYP and NADPH-CYP reductase cDNA inserts were obtained from Gentest Co. Enzyme Assays. 7-Ethoxycoumarin O-deethylation activities by CYP enzymes were determined by HPLC as described Incubation mixtures (final volume of 0.20 ml) for chlorzoxazone 6-hydroxylation were the same as for the assay of 7-Ethoxycoumarin Odeethylation, except that substrate was replaced by chlorzoxazone. Reactions were carried out at 37°C for 10 min and terminated by adding a mixture of 1.5 ml of CH 2 Cl 2 and 25 l of 43% H 3 PO 4 . The organic layer, after collecting by centrifugation, was evaporated to dryness under nitrogen atmosphere and the materials were dissolved in 200 l of 27% CH 3 CN containing 0.5% H 3 PO 4 . Product formation was determined by HPLC with a 4.6 ϫ 150 mm Nucleosil octylsilyl (C 8 ) reversed phase column (Chemco Scientific, Osaka, Japan). CYP contents were estimated spectrally by the original method Statistical Analysis. Kinetic parameters for 7-Ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation by human CYP enzymes were estimated with a computer program (KaleidaGraph; Synergy Software, Reading, PA) designed for nonlinear regression analysis. Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes Based on Kinetic Parameters of Recombinant CYP Enzymes. To define the potential roles of individual forms of CYP enzymes in the oxidation of 7-Ethoxycoumarin and chlorzoxazone by different human samples, we calculated the predicted activities of human liver microsomal 7-Ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation activities with kinetic parameters of recombinant human CYP enzymes and estimated contents of these CYP proteins by immunoblotting. The equation used for the prediction of expected activities was obtained from the previous method Results Kinetic Analysis of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by 14 Forms of Recombinant Human CYPs. Kinetic parameters for 7-Ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation by 14 forms of recombinant human CYP enzymes (in microsomes of T. ni cells) were determined as described in Materials and Methods K m values for 7-Ethoxycoumarin O-deethylation were the lowest in CYP1A1 followed by CYP1B1, CYP1A2, CYP2A6, CYP2E1, and CYP2C9 Of 14 forms of CYP examined, CYP2E1, 1A1, 2D6, 1A2, and 3A4 catalyzed chlorzoxazone 6-hydroxylation at varying rates, whereas other enzymes, including CYP1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 3A5, and 4A11 gave very low or undetectable rates for the hydroxylation Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes with Kinetic Parameters of Recombinant CYP Enzymes. We examined the 7-Ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation activities catalyzed by human liver microsomes with the kinetic parameters of recombinant human CYP enzymes described above and the estimated contents of immunochemically determined CYPs in human liver microsomes The levels of individual forms of CYP, including CYP1A2, CYP2A6, CYP2B6, CYP2C, CYP2D6, CYP2E1, and CYP3A in liver microsomes of 16 Japanese and 8 Caucasian samples were determined by Western immunoblotting analysi
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requirements for cytochrome b5 in the oxidation of 7 ethoxycoumarin chlorzoxazone aniline and n nitrosodimethylamine by recombinant cytochrome p450 2e1 and by human liver microsomes
Biochemical Pharmacology, 1996Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, L C Bell, F. Peter Guengerich, M. Nakano, Tsutomu ShimadaAbstract:NADH-dependent 7-Ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b(5) (b(5)), NADH-b(5) reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b(5) reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-Ethoxycoumarin O-deethylation activity, and further stimulation (similar to 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b(5) from the NADH- and NADPH-supported systems caused a 90% loss of 7-Ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. K-m values were increased and V-max values were decreased for 7-Ethoxycoumarin O-deethylation when b(5) was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b(5) in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-Ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline P-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-Ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b(5) immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b(5) is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b(5), and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.
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7 ethoxycoumarin o deethylation catalyzed by cytochromes p450 1a2 and 2e1 in human liver microsomes
Biochemical Pharmacology, 1996Co-Authors: Hiroshi Yamazaki, Mayumi Mimura, Peter F Guengerich, Kiyoshi Inoue, Tsutomu ShimadaAbstract:Abstract 7-Ethoxycoumarin O -deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O -deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-Ethoxycoumarin O -deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from membranes of Escherichia coli expressing modified human P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-Ethoxycoumarin O -deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O -deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low K m values with low V max values. Anti-human P450 2E1 antibodies inhibited markedly ( P O -deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 μM). However, we also found that anti-P450 1A2 antibodies suppressed O -deethylation activities only at a low substrate concentration (10 μM). Recombinant human P450 1A2 was found to have a low K m value for 7-Ethoxycoumarin O -deethylation, whereas P450 2E1 showed a high K m value. Of the P450 enzymes examined, P450 1A1 gave the highest O -deethylation activities with a low K m value, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O -deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a V max value similar to that of P450 1A2 and a K m value similar to that of P450 2E1. These results suggest that P450 1A2 is a low K m enzyme for 7-Ethoxycoumarin O -deethylation in human liver microsomes, although it has a lower V max value than P450 2E1.
Brian G Lake - One of the best experts on this subject based on the ideXlab platform.
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comparison of the metabolism of 7 ethoxycoumarin and coumarin in precision cut rat liver and lung slices
Food and Chemical Toxicology, 1995Co-Authors: R.j. Price, F Esclangon, P.t. Wield, J.a. Beamand, A B Renwick, David G Walters, Brian G LakeAbstract:Abstract The metabolism of 7-Ethoxycoumarin and [3- 14 C] coumarin was compared in precision-cut rat liver and lung slices. The lung slices were prepared using an agarose gel instilling technique enabling the production of tissue cylinders followed by lung slices employing a Krumdieck tissue slicer. Both 50μM 7-Ethoxycoumarin and 50μM [3- 14 C]coumarin were metabolized by rat liver and lung slices. 7-Ethoxycoumarin was converted to 7-hydroxycoumarin (7-HC) which was conjugated with both d -glucuronic acid and sulfate. 7-HC sulfate was the major metabolite formed by both liver and lung slices. [3- 14 C]Coumarin was metabolized by rat liver and lung slices to both polar products and to metabolite(s) that bound covalently to tissue slice proteins. The polar products included unidentified metabolites and 3-hydroxylation pathway products, with only very small quantities of 7-HC being formed. These results demonstrate that precision-cut lung slices area useful model in vitro system for studying the pulmonary metabolism of xenobiotics. Moreover, the precision-cut tissue slice technique may be employed for comparisons of hepatic and extrahepatic xenobiotic metabolism.
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metabolism of coumarin and 7 ethoxycoumarin by rat mouse guinea pig cynomolgus monkey and human precision cut liver slices
Xenobiotica, 1994Co-Authors: Aukje Steensma, J.a. Beamand, David G Walters, R.j. Price, Brian G LakeAbstract:1. The metabolism of 50 μM 7-Ethoxycoumarin and 50 μM [3-14C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man.2. In liver slices from all five species 7-Ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species.3. [3-14C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices we...
Kiyoshi Inoue - One of the best experts on this subject based on the ideXlab platform.
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roles of nadph p450 reductase in the o deethylation of 7 ethoxycoumarin by recombinant human cytochrome p450 1b1 variants in escherichia coli
Protein Expression and Purification, 2000Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi InoueAbstract:Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-Ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-Ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-Ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes, (C) 2000 Academic Press.
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Roles of NADPH-P450 reductase in the O-deethylation of 7-Ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli.
Protein Expression and Purification, 2000Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, F. Peter Guengerich, Kiyoshi InoueAbstract:Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-Ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-Ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-Ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was
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roles of nadph p450 reductase in the o deethylation of 7 ethoxycoumarin by recombinant human cytochrome p450 1b1 variants in escherichia coli
Protein Expression and Purification, 2000Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi InoueAbstract:Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-Ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-Ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-Ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.
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7 ethoxycoumarin o deethylation catalyzed by cytochromes p450 1a2 and 2e1 in human liver microsomes
Biochemical Pharmacology, 1996Co-Authors: Hiroshi Yamazaki, Mayumi Mimura, Peter F Guengerich, Kiyoshi Inoue, Tsutomu ShimadaAbstract:Abstract 7-Ethoxycoumarin O -deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O -deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-Ethoxycoumarin O -deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from membranes of Escherichia coli expressing modified human P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-Ethoxycoumarin O -deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O -deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low K m values with low V max values. Anti-human P450 2E1 antibodies inhibited markedly ( P O -deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 μM). However, we also found that anti-P450 1A2 antibodies suppressed O -deethylation activities only at a low substrate concentration (10 μM). Recombinant human P450 1A2 was found to have a low K m value for 7-Ethoxycoumarin O -deethylation, whereas P450 2E1 showed a high K m value. Of the P450 enzymes examined, P450 1A1 gave the highest O -deethylation activities with a low K m value, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O -deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a V max value similar to that of P450 1A2 and a K m value similar to that of P450 2E1. These results suggest that P450 1A2 is a low K m enzyme for 7-Ethoxycoumarin O -deethylation in human liver microsomes, although it has a lower V max value than P450 2E1.
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engineering bacterial cytochrome p450 p450 bm3 into a prototype with human p450 enzyme activity using indigo formation
Drug Metabolism and Disposition, 2010Co-Authors: Sunha Park, Heungchae JungAbstract:Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-Ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-Ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-Ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.
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generation of human metabolites of 7 ethoxycoumarin by bacterial cytochrome p450 bm3
Drug Metabolism and Disposition, 2008Co-Authors: Heungchae JungAbstract:Recently, wild-type and mutant forms of bacterial cytochrome P450 BM3 (CYP102A1) have been found to metabolize various drugs through reactions similar to those catalyzed by human cytochromes P450 (P450s). Therefore, it has been suggested that CYP102A1 may be used to produce large quantities of the metabolites of human P450-catalyzed reactions. In this report, we show that the oxidation of 7-Ethoxycoumarin, a typical human P450 substrate, is catalyzed by both wild-type and mutant forms of CYP102A1. Two major products were produced as a result of O -deethylation and 3-hydroxylation reactions. These results demonstrate that CYP102A1 mutants catalyze the same reactions as human P450s. High noncompetitive intermolecular kinetic deuterium isotope effects were observed for 7-Ethoxycoumarin O -deethylation in the CYP102A1 system. These results suggest that there is a common mechanism for the oxidation reactions catalyzed by both the bacterial CYP102A1 and human P450 enzymes.