(A) Recombinant Sphk2 activity as a function of brain sulfatide concentration. Activity was measured as described in experimental procedures. (B) Reaction rate as a function of substrate concentration for MBP-Sphk2, in the presence of 0 (■), 4 (○), 10 (X), or 25 (▼) μM sulfatide. Data points are the mean of two assays. Accompanying table shows Km and Vmax values calculated from the curves, using GraphPad PRISM software. Sulfatide produced a decrease in the enzyme turnover rate (Vmax), without a significant increase in substrate affinity (Km), indicative of non-competitive enzyme inhibition. (C) Inhibition of Cerk activity on C18:0 ceramide, by brain sulfatide incorporated into TritonX-100/cardiolipin/ceramide micelles at 5 or 50 μM (1.6% or 14.3% of micelle lipids). Results are the mean of two assays, and representative of two independent experiments.