Table 2

RSF-derived CPA and IFA 4-hydroxylase activities of different P-450 forms in HLS2

CYP forms:1A22A62B62C82C92C192D62E13A44A114-hydroxylase Activity
Liver microsomal P-450 Activity 2-a (measured)127538554522952707181373250489013802624822
Predicted 2-b
RSFCPA(Supersome)0.0122-c 0.064.8650.060.060.1050.00400.0230
Calculated 4-OH-CPA Activity1523021991816221011002736
% of total activity 2-d 0.68.480.
RSFCPA(Lymphoblast)0.050 2-c 0.0512.6280.0570.0510.235000.0210
Calculated 4-OH-CPA Activity31981186179040010301601
% of total activity 2-d 0.212.474.
Calculated 4-OH-IFA Activity01368813321001860456
% of total activity 2-d 029.819.32.770.10040.80
Calculated 4-OH-IFA Activity010015322182002060501
% of total activity 2-d
  • 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%.