TABLE 1.
Effect of reconstitution of multiple P450 enzymes on monooxygenase function: BPC vesicles
Reconstituted systems containing CPR plus CYP2B4 (CPR·2B4) or CPR plus CYP1A2 (CPR·1A2); a mixture of these two binary systems (CPR·1A2 + CPR·2B4); and a ternary system containing CPR, CYP1A2, and CYP2B4 (CPR·1A2·2B4) were incorporated into BPC membranes prepared using the DBB method and were used to measure the rate of metabolism of the listed substrates as described under “Experimental Procedures.” The abbreviations in the top row represent the components contained in the different reconstituted systems tested. When the components are separated by a chemical point, the enzymes were incorporated in the same lipid vesicles as a binary or ternary system. The plus sign is used to indicate the mixing of separate reconstituted systems immediately before performing the enzyme assays. The values represent the mean (expressed as nmol/min) ± S.D. of triplicate determinations. ND, not detected.
| Substrate | CPR·2B4 | CPR·1A2 | SUMa | CPR·1A2 + CPR·2B4 (change)b | CPR·1A2·2B4 (change) | 1A2 or 2B4 + CPR |
|---|---|---|---|---|---|---|
| nmol/min | nmol/min | nmol/min | nmol/min | nmol/min | nmol/min | |
| Luciferin-ME | ND | 0.2 ± 0.0 | 0.2 | 0.2 ± 0.0 (+11%) | 0.4 ± 0.1 (+105%) | NDc |
| 7ER | <0.1 | 0.3 ± 0.0 | 0.3 | 0.3 ± 0.0 (+0%) | 0.5 ± 0.0 (+66%) | <0.1c |
| 7EFC | 25.8 ± 2 | 0.8 ± 0.1 | 26.6 | 25.8 ± 0.3 (−3%) | 15.6 ± 0.6 (−41%) | <0.1d |
| Benzphetamine | 8.8 ± 0.4 | 0.7 ± 0.0 | 9.5 | 8.9 ± 0.4 (−6.3%) | 6.9 ± 0.3 (−27.5%) | 0.7 ± 0.0d |
| 7EC | 10.0 ± 0.1 | 0.5 ± 0.1 | 10.5 | 8.9 ± 0.6 (−15%) | 6.1 ± 0.3 (−42.1%) | 0.4 ± 0.3d |
| p-Nitroanisole | 1.2 ± 0.1 | 0.3 ± 0.0 | 1.3 | 1.1 ± 0.0 (−15.3%) | 0.7 ± 0.1 (−49.0%) | 0.2 ± 0.0d |
a The sum of the rates of the two binary systems containing CPR and one of the P450 enzymes are shown to indicate the rates expected if the two P450s did not interact with one another in the mixed systems.
b The numbers in parentheses represent the percentage change when compared with the sum of the binary systems. With all of the substrates, the rates of metabolism by the mixed binary and the ternary systems were statistically significant (p < 0.01) when analyzed by an unpaired t test.
c Metabolism was measured after mixing a simple reconstituted system of CYP1A2 in BPC with a simple system of CPR in BPC. This condition served as a negative control for CYP1A2-specific substrates to show that the CPR and P450 did not interact when in separate BPC vesicles.
d Metabolism was measured after mixing a simple reconstituted system of CYP2B4 in BPC with a simple system of CPR in BPC.