Protein | IgG | NADPH Oxidation | Testosterone 6β-Hydroxylation | Ratio of 6β-OH Testosterone Produced/NADPH Oxidized |
---|---|---|---|---|
nmol/min/nmol CYP3A4 | ||||
E. coli | − | 34.89 | 24.40 | 0.70 |
+ | 22.36 | 6.60 | 0.30 |
Microsomal protein from E. coli membranes expressing human CYP3A4 (0.05 nmol) and reductase was preincubated for 30 min at room temperature with 1.5 mg of purified IgG in 1 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 250 μM testosterone and 5 mM ascorbic acid. The sample was divided equally between two 0.50-ml cuvettes and the absorbance at 340 nm was measured. Reactions at 37°C were then initiated with NADPH (0.125 mM) and the absorbance was measured for 10 min at 37°C as described in Experimental Procedures. The reactions were stopped by the addition of 50 μl of 3 M HCl and 6β-hydroxytestosterone was quantified as described inExperimental Procedures. Values shown represent the mean of duplicate determinations, after the value of NADPH oxidation in the absence of testosterone had been subtracted from each value.