Kinetics of testosterone 6 beta-hydroxylation in the reconstituted system with similar ratios of purified CYP3A4, NADPH-cytochrome P450 oxidoreductase and cytochrome B-5 to human liver microsomes

Kinetics of testosterone 6 beta -hydroxylation were determined using a reco nstituted system that consisted of CYP3A4, cytochrome bs and NADPH-cytochro me P450 oxidoreductase (OR) with similar ratios as those seen in human live r microsomes and compared with those determined using human li ver microsom es. Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/C YP3A4. The K-m values of testosterone 6 beta -hydroxylation obtained from t he reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 3 5.2 muM, respectively, which were similar to that of the corresponding huma n liver microsomal samples (23.2 and 40.0 muM, respectively). However, V-ma x values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 a nd 31.1 pmol/min/pmol CYP3A4). The results suggest that the interaction bet ween substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolis m in the reconstituted systems is different from that in human liver micros omes. In conclusion, our reconstituted systems could be used for the determ ination of affinity but not for the determination of the maximum velocity o f substrate metabolism. Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b(5) and/or a specific lipid environment ar e required to establish a reconstituted system showing similar kinetic prop erties to those of human liver microsomes.