RSF-derived CPA and IFA 4-hydroxylase activities of different P-450 forms in HLS2
| CYP forms: | 1A2 | 2A6 | 2B6 | 2C8 | 2C9 | 2C19 | 2D6 | 2E1 | 3A4 | 4A11 | 4-hydroxylase Activity | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CPA | IFA | |||||||||||
| Measured | ||||||||||||
| Liver microsomal P-450 Activity 2-a (measured) | 1275 | 3855 | 452 | 295 | 2707 | 18 | 137 | 3250 | 4890 | 1380 | 2624 | 822 |
| Predicted 2-b | ||||||||||||
| RSFCPA(Supersome) | 0.0122-c | 0.06 | 4.865 | 0.06 | 0.06 | 0.105 | 0.004 | 0 | 0.023 | 0 | ||
| Calculated 4-OH-CPA Activity | 15 | 230 | 2199 | 18 | 162 | 2 | 1 | 0 | 110 | 0 | 2736 | |
| % of total activity 2-d | 0.6 | 8.4 | 80.4 | 0.7 | 5.9 | 0.1 | 0 | 0 | 4.0 | 0 | ||
| RSFCPA(Lymphoblast) | 0.050 2-c | 0.051 | 2.628 | 0.057 | 0.051 | 0.235 | 0 | 0 | 0.021 | 0 | ||
| Calculated 4-OH-CPA Activity | 3 | 198 | 1186 | 17 | 90 | 4 | 0 | 0 | 103 | 0 | 1601 | |
| % of total activity 2-d | 0.2 | 12.4 | 74.1 | 1.1 | 5.6 | 0.3 | 0 | 0 | 6.4 | 0 | ||
| RSFIFA(Supersome) | 0 | 0.026 | 0.195 | 0.042 | 0.019 | 0.032 | 0.002 | 0 | 0.038 | 0 | ||
| Calculated 4-OH-IFA Activity | 0 | 136 | 88 | 13 | 32 | 1 | 0 | 0 | 186 | 0 | 456 | |
| % of total activity 2-d | 0 | 29.8 | 19.3 | 2.7 | 7 | 0.1 | 0 | 0 | 40.8 | 0 | ||
| RSFIFA(Lymphoblast) | 0 | 0.026 | 0.338 | 0.074 | 0.01 | 0.098 | 0 | 0 | 0.042 | 0 | ||
| Calculated 4-OH-IFA Activity | 0 | 100 | 153 | 22 | 18 | 2 | 0 | 0 | 206 | 0 | 501 | |
| % of total activity 2-d | 0 | 20.0 | 30.5 | 4.4 | 3.6 | 0.4 | 0 | 0 | 41.1 | 0 | ||
↵2-a HLS2 liver microsomal P-450 profiling was performed with ten diagnostic P-450 reactions: phenacetin O-deethylase (1A2), coumarin 7-hydroxylase (2A6), 7-EFC O-deethylase (2B6), paclitaxel 6α-hydroxylase (2C8), diclofenac 4′-hydroxylase (2C9), (S)-mephenytoin 4′-hydroxylase (2C19), (+/−) bufuralol 1′-hydroxylase (2D6), p-nitrophenol hydroxylase (2E1), testosterone 6β-hydroxylase (3A4/5), and lauric acid ω-hydroxylase (4A11). Methods used for each of these P-450 activity assays are described by Chang, Crespi and Waxman in a series of ten chapters recently published (Phillips and Shephard, 1998). Activity data shown are in units of pmol product/min/mg microsomal protein. CPA and IFA 4-hydroxylase activities (2 mM substrate) were also measured as shown in the last two columns.
↵2-b Sum of calculated liver microsomal CPA and IFA 4-hydroxylase activities for each of the ten P-450 forms, determined using Eq 3. These values are in units of pmol/min/pmol P-450.
↵2-c RSF values were determined using phenacetinO-deethylase activity for Supersomes and 7-ethoxyresprufinO-deethylase activity for lymphoblasts (both serving as diagnostic reactions for CYP1A2). Accordingly, the RSF values shown for 1A2 cannot be directly compared for the two cDNA expression panels.
↵2-d Percentage of contribution of each P-450 form towards total calculated 4-hydroxylase activity, determined using Eq 4.; for example, for 2B6 using the RSFCPA (Supersomes) data, (calculated CPA 4-hydroxylase activity × 100)/(total predicted CPA 4-hydroxylase activity) = 2199/2736 = 80.4%.