RT Journal Article
SR Electronic
T1 Enhancement of Cytochrome P-450 3A4 Catalytic Activities by Cytochrome b
5 in Bacterial Membranes
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 999
OP 1004
VO 27
IS 9
A1 Hiroshi Yamazaki
A1 Miki Nakajima
A1 Mami Nakamura
A1 Satoru Asahi
A1 Noriaki Shimada
A1 Elizabeth M. J. Gillam
A1 F. Peter Guengerich
A1 Tsutomu Shimada
A1 Tsuyoshi Yokoi
YR 1999
UL http://dmd.aspetjournals.org/content/27/9/999.abstract
AB 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. The American Society for Pharmacology and Experimental Therapeutics