Fig. 3.
SphK2 activity-dependent changes in NBD-Sph fluorescence emission. A: λEx = 550 nm fluorescence emission spectra of NBD-Sph (75 µM) at 25°C in SphK2 reaction buffer containing 0.07% Triton X-100, 200 mM KCl, and 13.8 nM SphK2, but no ATP. Inset: Time-resolved fluorescence emission with λEx = 550 nm and λEm = 583 nm in the absence or presence of ATP or SphK2 as indicated. B: Time-resolved fluorescence emission of NBD-Sph (75 µM) at 37°C in SphK2 reaction buffer containing 0.07% Triton X-100, 200 mM KCl, 1 mM ATP, and increasing SphK2 concentrations (n = 3). Inset: Initial rates versus [SphK2] plot. C: Time-resolved fluorescence emission at 37°C of solutions containing 0.1% Triton X-100, 200 mM KCl, 1 mM ATP, 6.9 nM SphK2, and increasing NBD-Sph concentrations. n = 3. Inset: Initial rates versus [NBD-Sph] plot. Experiments in B and C were performed in 384-well 20 µl format in a fluorescence plate reader. Fluorescence emission was measured with λEx = 550 nm and λEm= 583 nm.