Figure 1. Purification of (NH₄)₂SO₄-extractable, membrane-associated, Mg²⁺-dependent neutral SMase from bovine brain.

A membrane-associated neutral form of N-SMase was extracted with 0.2 M (NH₄)₂SO₄ from 10,000× g pellets of bovine brain homogenates and purified by sequential chromatography steps. (A) Mono S cationic exchange FPLC and peak II active fractions were applied to Superose 12 gel filtration chromatography. (B) The active fraction purified from Superose 12 gel filtration chromatography was analyzed using two-dimensional gel electrophoresis. The results from this purification scheme are representative of 10 independent experiments.