Figure 7.

FTY720 reduces the enzymatic activity of recombinant cPLA₂α. (A) Recombinant cPLA₂α (0.5 μg) activity was measured in the absence or presence of 4 mol% FTY720 or C1P, or 2 mol% of each in a mixed micelle assay. Data are means ± SD of duplicate determinations expressed as nanomole AA released per minute per milligram. (B) Stoichiometry of cPLA₂ inhibition by FTY720. Recombinant cPLA₂α (0.5 μg) activity was measured in the absence or presence of increasing mol% FTY720 (100 × [FTY720]/[Triton X-100 + PC + FTY720]). The PC concentration was fixed at 15 mol% (100 × [PC]/[Triton X-100 + PC + FTY720]). Data are means ± SD of duplicate determinations expressed as nanomole AA produced per minute per milligram. Nonlinear regression analysis was performed (R² = 0.999). The IC₅₀ value for FTY720 inhibition of cPLA₂α activity was 1.1 ± 0.03 mol%, and the Hill coefficient was 2.3 ± 0.1. (C) Lack of effect of sphingoid bases on cPLA₂α activity. Recombinant cPLA₂ (0.5 μg) activity was determined in the absence or presence of 4 mol% FTY720, FTY720-P, sphingosine, or S1P. Data are expressed as fold change in cPLA₂α activity compared with control activity (none) and are means ± SD of triplicate determinations. Similar results were obtained in 3 independent experiments.