Figure 7.

Lack of 3HCIM specific binding to rCYP2C11. Left: Sf9 microsomes expressing rCYP2C11 (0.5 or 2.0 pmol enzyme [1.35 or 5.4 μg protein, respectively] per tube, or nontransfected microsomes (control, 10 μg protein) were incubated with 3HCIM (50 nM) and filtered as in Figure 3. 3HCIM binding (left ordinate, mean ± SEM from triplicate determinations) is shown from a single experiment. Right: To ensure that CYP2C11 was properly filtered by the binding assay, CYP2C11 (50 pmol enzyme, 135 μg protein) or liver membranes (100,000 x g pellet, 387 μg) were incubated as in the left panel but in the absence of 3HCIM. Samples were filtered with either filter paper present or absent, followed by washing as described, or not filtered at all (100% controls). Filtrates or control samples (860 μl) were analyzed for CYP2C11 activity as described. Control enzyme activities of the unflitered CYP2C11 sample (13.5 μg protein) and the unfiltered liver microsome sample (38.7 μg protein) were 5.7 nmol/min-mg and 1.3 nmol/min-mg, respectively. Right ordinate shows percent of control enzyme activity detected (mean ± SEM) of 2-3 determinations from one experiment. These experiments show that rCYP2C11 is viable (right), is filtered in the radioligand-binding assay (right), but does not specifically bind 3HCIM (left).