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Tsutomu Shimada - One of the best experts on this subject based on the ideXlab platform.

  • 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, 2000
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, F. Peter Guengerich, Kiyoshi Inoue
    Abstract:

    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

  • 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, 2000
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi Inoue
    Abstract:

    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.

  • 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, 2000
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi Inoue
    Abstract:

    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.

  • prediction of human liver microsomal oxidations of 7 Ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome p 450 enzymes
    Drug Metabolism and Disposition, 1999
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi Yamazaki
    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.

  • 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, 1999
    Co-Authors: Hiroshi Yamazaki, Masaaki Tanaka, Tsutomu Shimada
    Abstract:

    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.

  • prediction of human liver microsomal oxidations of 7 Ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome p 450 enzymes
    Drug Metabolism and Disposition, 1999
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi Yamazaki
    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.

  • 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, 1999
    Co-Authors: Hiroshi Yamazaki, Masaaki Tanaka, Tsutomu Shimada
    Abstract:

    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.

  • 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, 1996
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, L C Bell, F. Peter Guengerich, M. Nakano, Tsutomu Shimada
    Abstract:

    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.

  • 7 Ethoxycoumarin o deethylation catalyzed by cytochromes p450 1a2 and 2e1 in human liver microsomes
    Biochemical Pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Mayumi Mimura, Peter F Guengerich, Kiyoshi Inoue, Tsutomu Shimada
    Abstract:

    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.

  • 7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.
    Biochemical pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Mayumi Mimura, F. Peter Guengerich, Kiyoshi Inoue, Yoshimitsu Oda, Tsutomu Shimada
    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 the membrane of Escherichia coli expressing modified 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 Km values with low Vmax values. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-Ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM). Recombinant human P450 1A2 was found to have a low Km value for 7-Ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km 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 Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1. These results suggest that P450 1A2 is a low Km enzyme for 7-Ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1.

Kiyoshi Inoue - One of the best experts on this subject based on the ideXlab platform.

  • 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, 2000
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi Inoue
    Abstract:

    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.

  • 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, 2000
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, F. Peter Guengerich, Kiyoshi Inoue
    Abstract:

    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

  • 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, 2000
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Elizabeth M J Gillam, Peter F Guengerich, Kiyoshi Inoue
    Abstract:

    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.

  • 7 Ethoxycoumarin o deethylation catalyzed by cytochromes p450 1a2 and 2e1 in human liver microsomes
    Biochemical Pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Mayumi Mimura, Peter F Guengerich, Kiyoshi Inoue, Tsutomu Shimada
    Abstract:

    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.

  • 7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.
    Biochemical pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Mayumi Mimura, F. Peter Guengerich, Kiyoshi Inoue, Yoshimitsu Oda, Tsutomu Shimada
    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 the membrane of Escherichia coli expressing modified 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 Km values with low Vmax values. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-Ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM). Recombinant human P450 1A2 was found to have a low Km value for 7-Ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km 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 Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1. These results suggest that P450 1A2 is a low Km enzyme for 7-Ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1.

Taejin Oh - One of the best experts on this subject based on the ideXlab platform.

  • homology modeling and in vitro analysis for characterization of streptomyces peucetius cyp157c4
    Journal of Microbiology and Biotechnology, 2015
    Co-Authors: Hemraj Rimal, Jong Hwa Jang, Sangcheol Yu, Taejin Oh
    Abstract:

    : In this study, we tried to characterize Streptomyces peucetius CYP157C4 with homology modeling using three cytochrome P450 (CYP) structures (CYP157C1, CYP164A2, and CYP107L1), having discovered that CYP157C4 lacks the ExxR motif that was considered invariant in all CYPs. We used Discovery Studio 3.5 to build our model after first assessing the stereochemical quality and side-chain environment, and a 7-Ethoxycoumarin substrate was docked into the final model. The model-substrate complex allowed us to identify functionally important residues and validate the active-site architecture. We found a distance of 4.56 A between the 7-Ethoxycoumarin and the active site of the heme, and cloning and an in vitro assay of the CYP157C4 showed the dealkylation of the substrate. Since the details regarding this group of CYP structures are still unknown, the findings of this study may provide elucidation to assist with future efforts to find a legitimate substrate.

  • hydrogen peroxide mediated dealkylation of 7 Ethoxycoumarin by cytochrome p450 cyp107aj1 from streptomyces peucetius atcc27952
    Enzyme and Microbial Technology, 2011
    Co-Authors: Narayan Prasad Niraula, Bashistha Kumar Kanth, Jae Kyung Sohng, Taejin Oh
    Abstract:

    Cytochrome P450 CYP107AJ1, which was isolated from Streptomyces peucetius and showed high homology with peroxygenases, catalyzed a dealkylation reaction with hydrogen peroxide to provide electrons, protons and oxygen, evading the requirement for a supporting redox protein. Preliminary investigation of its transcriptional level in S. peucetius showed significant expression. Homology modeling and subsequent docking with 7-Ethoxycoumarin yielded a reasonable docked structure. cyp107AJ1 cloned into pET28a(+) was expressed in Escherichia coli, and soluble protein was subjected to column-chromatographic purification in order to carry out enzyme assays with 7-Ethoxycoumarin. HPLC analysis of the extracted product, corresponding to its LC/MS analysis, showed the dealkylated 7-Ethoxycoumarin, which was further established by subsequent GC/MS spectral analysis. We suggest that CYP107AJ1 bypassed the requirement for NAD(P)H and redox partners for generating novel analogues.

Brian G Lake - One of the best experts on this subject based on the ideXlab platform.

  • Influence of Slice Thickness and Culture Conditions on the Metabolism of 7-Ethoxycoumarin in Precision-cut Rat Liver Slices.
    Alternatives to laboratory animals : ATLA, 1998
    Co-Authors: R.j. Price, A B Renwick, J. B. Houston, P. T. Barton, Brian G Lake
    Abstract:

    This study investigated the effects of some experimental variables on the rate of xenobiotic metabolism in precision-cut rat liver slices. Liver slices of 123 ± 8μm (mean ± SEM of six slices), 165 ± 3μm, 238 ± 6μm and 515 ± 14μm thickness were prepared from male Sprague-Dawley rats, and incubated in RPMI 1640 medium in an atmosphere of 95% O2/5% CO2 by using a dynamic organ culture system. Liver slices of all thicknesses metabolised 10μM 7-Ethoxycoumarin to total (free and conjugated) 7-hydroxycoumarin in a time-dependent manner. The rate of 7-Ethoxycoumarin metabolism was greatest in 165μm thick slices and slowest in 515μm thick slices, being 2.74 ± 0.19pmol/minute/mg slice protein and 0.69 ± 0.07pmol/minute/mg slice protein, respectively. No marked effects on the rate of 7-Ethoxycoumarin metabolism in liver slices were observed either by changing the medium to Earle's balanced salt solution (EBSS) or by changing the gas phase to 95% air/5% CO2. Moreover, the perfusion of rat livers with EBSS at 2–4°C, prior to preparation of tissue cores, did not enhance 7-Ethoxycoumarin metabolism in rat liver slices. In this study, the optimal slice thickness was 175μm, with higher rates of 7-Ethoxycoumarin metabolism being observed than with 250μm thick slices, which are often used for studies of xenobiotic metabolism. Variable results were obtained with slices of around 100–120μm thickness, which may be attributable to the ratio between intact hepatocytes and cells damaged by the slicing procedure in these very thin slices.

  • comparison of the metabolism of 7 Ethoxycoumarin and coumarin in precision cut rat liver and lung slices
    Food and Chemical Toxicology, 1995
    Co-Authors: R.j. Price, F Esclangon, P.t. Wield, J.a. Beamand, A B Renwick, David G Walters, Brian G Lake
    Abstract:

    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.

  • metabolism of coumarin and 7 Ethoxycoumarin by rat mouse guinea pig cynomolgus monkey and human precision cut liver slices
    Xenobiotica, 1994
    Co-Authors: Aukje Steensma, J.a. Beamand, David G Walters, R.j. Price, Brian G Lake
    Abstract:

    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...