Fig. 1. Characterization of SMase activities in hippocampal neurons. A, Cation dependence. Homogenates of hippocampal neurons (preplated) (20 μg of protein) were assayed in 25 mm KCl, and 50 mm Tris, pH 7.4, for N-SMase activity or in MES buffer, pH 4.7, for A-SMase activity, by incubation with C6-NBD-SM (10 μm) in a final volume of 200 μl, with or without addition of cations or EDTA. After 1 hr at 37°C, the reaction was terminated, and C6-NBD-Cer formation was analyzed. Results are means ± SEM of three independent experiments. B, Specific activity. Homogenates of hippocampal neurons (20 μg of protein) were assayed in 25 mm KCl, 10 mm MgCl2, and 50 mm Tris, pH 7.4, for N-SMase activity or in MES buffer, pH 4.7, for A-SMase activity, by incubation with C6-NBD-SM in a final volume of 50 μl. After 1 hr at 37°C, the reaction was terminated, and C6-NBD-Cer formation was analyzed. Results are means ± SEM of two independent experiments.