TABLE 2.
RP-HPLC analysis of the extent of AA oxygenation at a high ratio of huPGHS-1 to AA in the presence or absence of unlabeled AA, PA, or OA
| Fatty Acid Added Four Min After Initiating the Reaction | Unreacted [1-14C]Arachidonic Acid Remaining After 8 min (Ave. ±SD from Three Reactions(% of Starting Radioactivity) |
| Control (no FA added) | 11 ± 1.2ab |
| 60 μM AA | 2.4 ± 0.06ac |
| 1 μM PA | 8.7 ± 1.7c |
| 5 μM PA | 6.4 ± 1.2c |
| 25 μM PA | 3.0 ± 0.90c |
| 2 μM OA | 3.9 ± 0.72c |
| 10 μM OA | 2.9 ± 1.1b,c |
[1-14C]AA (1 μM) was incubated with huPGHS-1 (1 μM) at 37°C for 4 min, then unlabeled AA, PA, or OA 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-RP-HPLC to separate the radioactive products and unreacted AA, as described in Experimental Procedures. The results are shown as the percentage of 14C label remaining in the HPLC fraction eluting with unreacted AA.
Value is from Table 1.
Value shown is average ± SD for six determinations, and other values shown are average ± SD for triplicate determinations.
Significant difference from the control value (no FA added at 4 min) in Student t-test (P < 0.05).