Article Figures & Data
Figures
- Fig. 1.
Detection of physical complexes between CYP1A2 and CYP2B4 using BRET. (A) Physical complex formation between CYP2B4 and CYP1A2. pGFP2-N1/CYP2B4-GFP and pRluc-N2/CYP1A2-Rluc were transfected into HEK-293T/17 cells at different ratios and BRET was measured. (B) Effect of POR on the CYP1A2/CYP2B4 BRET pair. pGFP2-N1/CYP1A2-GFP and pRluc-N2/CYP2B4-Rluc were cotransfected into HEK-293T/17 cells (blue). This was repeated in the presence of cotransfected unlabeled POR, which did not measurably affect BRETmax (red), indicating that the CYP1A2-CYP2B4 complex is stable in the presence of POR. The mean ± S.D. value for triplicate determinations is represented unless the error bars are smaller than the plotted points.
- Fig. 2.
Effect of unlabeled CYP1A2 on the POR/CYP2B4 BRET pair. pGFP2-N1/CYP2B4-GFP and pRluc-N1/POR-Rluc were cotransfected into HEK-293T/17 cells at varying GFP:Rluc ratios both in the absence (blue) and presence (red) of cotransfected, unlabeled CYP1A2. Each point represents the mean of triplicate determinations with the S.D. value within the data point.
- Fig. 3.
Effect of unlabeled CYP2B4 on the BRET-sensitive POR-CYP1A2 complex. pGFP2-N1/CYP1A2-GFP and pRluc-N1/POR-Rluc were cotransfected into HEK-293T/17 cells at varying GFP:Rluc ratios in the absence (blue) and presence (red) of cotransfected unlabeled CYP2B4. Points represent the mean ± S.D. values for three measurements; for most points, the error is smaller than the data point.
- Fig. 4.
Effect of cotransfection of CYP1A2-GFP and CYP2B4-GFP on PROD and EROD in microsomes isolated from transfected HEK-293T/17 cells. Cells were transfected with pGFP2-N1/CYP1A2-GFP, pGFP2-N1/CYP2B4-GFP, or both. Microsomes derived from these cells were then incubated in the presence of either 7-pentoxyresorufin or 7-ethoxyresorufin, and the initial rate of resorufin accumulation was determined spectrofluorometrically. The experimentally determined rates were then compared with those predicted for CYP1A2 and CYP2B4 if they behaved as monomers that simply competed for the available POR. Because each group of transfected cells contained different levels of POR, CYP1A2, and CYP2B4, the predicted rates were determined using Dynafit 4, based on the model described in Supplemental Fig. 1. Consequently, if the P450 enzymes affected each other’s function, the experimental data would be expected to deviate from the “predicted” activities. This analysis showed that cotransfection of both P450s led to lower rates of PROD activity (A) and higher rates of EROD activity (B) than predicted by simple competition. Protein concentrations from the CYP1A2 group were 0.048 and 0.005 μM for CYP1A2 and POR, respectively. Concentrations for the CYP2B4 group were 0.040 and 0.005 μM for CYP2B4 and POR, respectively. Concentrations for the group cotransfected with both CYP1A2 and CYP2B4 were 0.051, 0.045, and 0.005 μM for CYP1A2, CYP2B4, and POR, respectively. The activities represent the mean ± S.E.M. values for four replicates. Unpaired t test: *P < 0.05; ***P < 0.001 for the difference between the measured and predicted values.
- Fig. 5.
Effect of unlabeled POR on the BRET signal for the CYP1A2-CYP1A2 complex. pGFP2-N1/CYP1A2-GFP and pRluc-N2/CYP1A2-Rluc were cotransfected into HEK-293T/17 cells, and BRET was measured (blue curve). This was repeated in the presence of cotransfected unlabeled POR (red curve). The decrease in BRETmax is consistent with disruption of the CYP1A2-CYP1A2 complex. Each point represents the mean value of triplicate determinations with the S.D. value within the data points.
- Fig. 6.
Effect of unlabeled CYP2B4 on the BRET signal for the CYP2B4-CYP2B4 BRET pair. pGFP2-N1/CYP2B4-GFP and pRluc-N2/CYP2B4-Rluc were cotransfected into HEK-293T/17 cells, and BRET was measured in the presence and absence of different amounts of cotransfected unlabeled CYP2B4. The presence of unlabeled competitor did not have an effect on the BRETmax, suggesting that CYP2B4-GFP and CYP2B4-Rluc do not form a specific BRET-detectible complex. Each point represents the mean value of triplicate determinations with the S.D. value within the data points.
Additional Files
Data Supplement
- Supplemental Data -
Supplemental Table 1 – Information on the GFP, Rluc, and WT P450 System Protein Constructs
Supplemental Figure 1 – Dynafit 4 script used to predict both 7-ethoxyresorufin-Odealkylation (EROD) and 7-pentoxyresorufin-O-dealkylation (PROD) when CYP1A2 and CYP2B4 exist as functional monomers that simply compete for limiting concentrations of NADPH-cytochrome P450 reductase (CPR)
- Supplemental Data -
In this issue
Specific Complexes among CYP1A2, CYP2B4, and P450 Reductase
