Protein | System | IgG | Testosterone 6β-Hydroxylation | Relative Activity |
---|---|---|---|---|
nmol/min/nmol 3A4 | % | |||
Human liver microsomes | Cumene | − | 2.36 | 100 |
Hydroperoxide | + | 0.11 | 5 | |
NADPH | − | 10.18 | 100 | |
+ | 0.28 | 3 | ||
E. coli (+Red) | Cumene | − | 4.54 | 100 |
Hydroperoxide | + | 0.23 | 5 | |
NADPH | − | 28.70 | 100 | |
+ | 2.46 | 9 | ||
E. coli (−Red) | Cumene | − | 5.12 | 100 |
Hydroperoxide | + | 0.19 | 4 | |
NADPH | − | N.D. | N.D. | |
+ | N.D. | N.D. |
Microsomal protein from human liver (75 μg; calculated to correspond to 10 pmol CYP3A4, when CYP3A4 is considered to comprise 30% of total P450 in the sample), microsomal protein from E. coliexpressing human CYP3A4 (10 pmol) and NADPH-P450 reductase (+Red), or microsomal protein from E. coli expressing only human CYP3A4 (10 pmol) (−Red) was preincubated for 30 min at room temperature with 0.7 mg of purified IgG. The reaction was then started by the addition of 250 μM testosterone and 2 mM NADPH or 200 μM of cumene hydroperoxide and incubated for 12.5 min at 37°C. Reactions were terminated by the addition of 1.5 ml of dichloromethane and the amount of 6β-hydroxytestosterone produced was determined as described inExperimental Procedures. Values represent the mean of duplicate determinations.
N.D., not determined.