Identification of multiple forms of membrane-associated neutral sphingomyelinase in bovine brain

Abstract

Many different stimuli such as bioactive agents and environmental stresses are known to cause the activation of sphingomyelinase (SMase), which hydrolyzes sphingomyelin to generate ceramide as a second messenger playing a key role in differentiation and apoptosis in various cell types. Here we identified multiple forms of the membrane-associated neutral SMase (N-mSMase) activity in bovine brain. They could be classified into two groups according to extracting agents: group T-mSMase, extracted with 0.2% Triton X-100, and group S-mSMase, extracted with 0.5 M (NH4)(2)SO4. Group T-mSMase was further separated into four forms of T-mSMase: alpha, beta, gamma, and delta, which were extensively purified from 40,000-g pellets of bovine brain homogenates by 3,150-, 5,275-, 1,665- and 2,556-fold over the membrane extracts, respectively, by sequential use of several column chromatographies. On the other hand, S-mSMase was eluted as two active peaks of S-mSMase epsilon and zeta in a phenyl-5PW hydrophobic HPLC column and further purified by 1,119- and 976-fold over 40,000-g pellets of the homogenates, respectively. These highly purified N-mSMase enzyme preparations migrated as several bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed many different features in biochemical properties such as pH dependence, Mg2+ requirements, and effects of detergents. Taken together, our data strongly suggest that mammalian brain N-mSMase may exist as multiple forms different in both its chromatographic profiles and biochemical properties.