FIGURE 5.

Kinetic analysis of CerS4 inhibition by FTY 720. Homogenates (100 μg of protein) were prepared from HEK cells overexpressing CerS4 and incubated with (squares) or without (circles) 25 μm FTY720. A, CerS4 activity was assayed using 50 μm C18-CoA and increasing amounts of [4,5-³H]sphinganine/sphinganine, 20 μm defatted bovine serum albumin (such that the ratio of [4,5-³H]sphinganine to sphinganine was the same at each sphinganine concentration). The data were plotted according to Lineweaver-Burk. The slopes of the lines were parallel, with values of 0.0169 ± 0.0019 and 0.0167 ± 0.0019 with and without FTY720, respectively, demonstrating that FTY inhibits CerS4 activity toward sphinganine via uncompetitive inhibition. The results are shown for a typical experiment repeated three times. B, CerS4 activity was assayed using 0.25 μCi of [4,5-³H]sphinganine, 15 μm sphinganine, 20 μm defatted bovine serum albumin, and increasing amounts of C18-acyl-CoA. The data were plotted according to the best fit of a sigmoid dose-response curve, which gave r² values of 0.99 for both curves, and demonstrate that FTY720 inhibits CerS4 via noncompetitive inhibition toward C18-acyl-CoA. The inset shows a Lineweaver-Burk plot from a different experiment in which defatted bovine serum albumin was not included in the reaction buffer. Noncompetitive inhibition toward C18-acyl-CoA is also observed. The results are the means ± S.D. from two independent experiments repeated five or six times.