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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.
Biochem Pharmacol. 1996 Jul 26; 52(2):301-9.BP

Abstract

NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 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-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 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. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 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 b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 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 b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

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Publisher Full Text

Authors+Show Affiliations

Yamazaki H
Osaka Prefectural Institute of Public Health, Japan.
Nakano M
No affiliation info available
Gillam EM
No affiliation info available
Bell LC
No affiliation info available
Guengerich FP
No affiliation info available
Shimada T
No affiliation info available

MeSH

Aniline CompoundsChlorzoxazoneCoumarinsCytochrome P-450 CYP1A2Cytochrome P-450 CYP2E1Cytochrome P-450 Enzyme InhibitorsCytochrome P-450 Enzyme SystemCytochromes b5DimethylnitrosamineHumansKineticsMicrosomes, LiverNADH, NADPH OxidoreductasesNADPH-Ferrihemoprotein ReductaseOxidation-ReductionOxidoreductasesOxidoreductases, N-DemethylatingRecombinant Proteins

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8694855

Citation

Yamazaki, H, et al. "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, vol. 52, no. 2, 1996, pp. 301-9.
Yamazaki H, Nakano M, Gillam EM, et al. 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. Biochem Pharmacol. 1996;52(2):301-9.
Yamazaki, H., Nakano, M., Gillam, E. M., Bell, L. C., Guengerich, F. P., & Shimada, T. (1996). 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, 52(2), 301-9.
Yamazaki H, et al. 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. Biochem Pharmacol. 1996 Jul 26;52(2):301-9. PubMed PMID: 8694855.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Yamazaki,H, AU - Nakano,M, AU - Gillam,E M, AU - Bell,L C, AU - Guengerich,F P, AU - Shimada,T, PY - 1996/7/26/pubmed PY - 1996/7/26/medline PY - 1996/7/26/entrez SP - 301 EP - 9 JF - Biochemical pharmacology JO - Biochem Pharmacol VL - 52 IS - 2 N2 - NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 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-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 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. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 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 b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 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 b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes. SN - 0006-2952 UR - https://www.unboundmedicine.com/medline/citation/8694855/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0006-2952(96)00208-0 DB - PRIME DP - Unbound Medicine ER -