Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.
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 coli transformed 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 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-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 b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) 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 containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.
Yamazaki
H
H
Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan. yamazak@kenroku.kanazawa-u.ac.jp
Nakajima
M
M
Nakamura
M
M
Asahi
S
S
Shimada
N
N
Gillam
E M
EM
Guengerich
F P
FP
Shimada
T
T
Yokoi
T
T
D018686:Anesthetics, Intravenous / Q000378:metabolism; D001419:Bacteria / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009543:Nifedipine / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
D018686:Anesthetics, Intravenous; D002121:Calcium Channel Blockers; D011994:Recombinant Proteins; D013739:Testosterone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; D009543:Nifedipine; D008874:Midazolam