Search

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-mSM-ase, extracted with 0.2% Triton X-100, and group S-mSMase, extracted with 0.5 M (NH 4 ) 2 SO 4 . Group T-mSMase was further separated into four forms of T-mSMase: a, β, γ, and δ, 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 ∈ and ζ 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, Mg 2+ 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.

A convenient and rapid method for the oxidation of α-methylene ketones to α-diketones has been described involving the reaction of pyridine N-oxide with α-nosyloxy ketone intermediates.