Roles of cytochrome b5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes.
NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-hydroxylation activities in the presence of NADPH-P450 reductase but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH-P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 affinity chromatography) inhibited testosterone 6 beta-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH-P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2 stimulated rates of reduction of b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH-P450 reductase are accelerated by complexation with substrates and b5 is supported in this study.
Yamazaki
H
H
Osaka Prefectural Institute of Public Health, Japan.
Nakano
M
M
Imai
Y
Y
Ueng
Y F
YF
Guengerich
F P
FP
Shimada
T
T
D000818:Animals; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009543:Nifedipine / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism*
D011994:Recombinant Proteins; D015636:Magnesium Chloride; D009243:NAD; D013739:Testosterone; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D009543:Nifedipine