Purification of neutral sphingomyelinase 2 from bovine brain and its calcium-dependent activation.

Ceramide is produced by sphingomyelinase (SMase) and it plays a key role in cellular responses such as apoptosis. In this study, we report the purification and characterization of neutral SMase2 (nSMase2) from bovine brain tissue. Triton X-100 extracts of bovine brain membranes were purified in nine steps, including sequential chromatography. The specific activity of purified nSMase increased 8183-fold over the brain membrane fraction. Purified nSMase showed similarities to nSMase2, which had been purified and cloned previously. Interestingly, purified nSMase2 was Ca2+-dependent and could be activated by micromolar concentrations of Ca2+ under Mg2+-free conditions. Ceramide generation was dependent upon the calcium ionophore A23187 and was observed in nSMase2-over-expressing COS-7 cells. This generation was suppressed by GW4869, an nSMase2 inhibitor, but not to fumonisin B(1), an inhibitor of the de novo ceramide synthesis pathway. The present study demonstrates the Ca2+-dependent activation of nSMase2.