Abstract
Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia colitransformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6β-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b5(b5) and apolipoproteinb5 further enhanced the testosterone 6β-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b5). Apparent maximal activities of testosterone 6β-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b5 was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6β-hydroxylation, nifedipine oxidation, and midazolam 4- and 1′-hydroxylation activities in CYP3A4/NPR membranes plusb5 systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containingb5 are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.
Footnotes
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Send reprint requests to: Hiroshi Yamazaki, Ph.D., Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail: yamazak{at}kenroku.kanazawa-u.ac.jp
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This work was supported in part by grants from the Japanese Ministries of Education, Science, Sports, and Cultures; and Health and Welfare.
- Abbreviations used are::
- P-450
- cytochrome P-450
- NPR
- NADPH-P-450 reductase
- CYP3A4/NPR membranes
- membranes prepared from bacteria coexpressing CYP3A4 and NPR from a bicistronic vector
- apo b5
- apolipoprotein cytochromeb5
- LB
- Luria-Bertani medium
- TB
- Terrific Broth
- Received March 9, 1999.
- Accepted June 2, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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