TABLE 3.
RP-HPLC analysis of the displacement of AA from Eallo of huPGHS-2 by unlabeled DHA, DPA, adrenic acid, linoleic acid, or PA
[1-14C]AA (1 μm) was incubated with huPGHS-2 (1 μm) at 37 °C for 4 min and then the indicated amounts of an unlabeled FA was added, and the incubation was continued for another 4 min. The reactions were stopped by the addition of ethyl acetate/acetic acid (20:1), and an aliquot of the organic phase was subjected to radio-reverse phase-HPLC to separate the radioactive products and unreacted AA as described under “Experimental Procedures.” The results are shown as the percentage of total 14C label that remained in the RP-HPLC fraction co-eluting with unreacted AA.
| Fatty acid added 4 min after initiating the reaction | Unreacted [1-14C]arachidonic acid remaining after 8 min (average ± S.E. from 2 reactions with replicates in parentheses) (% of starting radioactivity)a |
|---|---|
| Control (no FA added) | 13.5 ± 2.4 (11.8, 15.2) |
| 2.0 μm DHA | 11.1 ± 0.67 (11.6, 10.7) |
| 5.0 μm DHA | 11.0 ± 2.50 (12.8, 9.3) |
| 10 μm DHA | 11.3 ± 1.25 (10.4, 12.2) |
| 20 μm DHA | 8.84 ± .98 (8.2, 9.5) |
| 2.0 μm DPA | 14.0 ± 2.91 (11.9, 16.1) |
| 5.0 μm DPA | 11.1 ± 2.69 (13.0, 9.2) |
| 10 μm DPA | 10.7 ± 1.88 (9.4, 12.0) |
| 20 μm DPA | 7.44 ± 0.42 (7.7, 7.1) |
| 5 μm PA | 1.48 ± 0.28b (1.7, 1.3) |
| Control (no FA added) | 10.6 ± 1.38 (9.6, 11.5) |
| 2.0 μm adrenic acid | 12.8 ± 0.54 (12.5, 13.2) |
| 5.0 μm adrenic acid | 9.31 ± 3.32 (7.0, 11.7) |
| 10 μm adrenic acid | 7.96 ± 0.42 (7.7, 8.3) |
| 20 μm adrenic acid | 7.46 ± 0.22 (7.6, 7.3) |
| 2.0 μm linoleic acid | 7.49 ± 0.74 (8.0, 7.0) |
| 5.0 μm linoleic acid | 6.27 ± 0.02c (6.3, 6.3) |
| 10 μm linoleic acid | 4.69 ± 0.06c (4.6, 4.7) |
| 20 μm linoleic acid | 1.37 ± 1.28c (2.3, 0.5) |
a Value shown is average ± S.E. for duplicate determinations from one experiment representative of two separate experiments performed at different times with different enzyme preparations; replicates are in parentheses.
b Data are significantly different from the control value (13.5 ± 2.4%) in Student's t test (p < 0.05).
c Data are significantly different from the control value (10.6 ± 1.38%) in Student's t test (p < 0.05).