Identification of Multiple Forms of Membrane‐Associated Neutral Sphingomyelinase in Bovine Brain

Abstract

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 (NH 4 ) 2 SO 4 . Group TmSMase was further separated into four forms of TmSMase: ␣, ␤, ␥, and ␦, 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 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 NmSMase 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 2ϩ 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. Key Words: Neutral sphingomyelinase -Ceramide -PurificationMammalian brain.

Mammalian Neutral Sphingomyelinases: Regulation and Roles in Cell Signaling Responses

“…Together, these enzymes comprise the N-SMase family. Notably, the existence of multiple nSMase isoforms is in agreement with studies reporting multiple N-SMase activities reported in bovine brain (Jung et al, 2000). …”

Ceramide and neurodegeneration: Susceptibility of neurons and oligodendrocytes to cell damage and death

“…Biochemical characterization of this NSMase revealed that it is sensitive to glutathione [127]. Multiple forms of the membrane-bound NSMases have been identified in bovine brain [128]. This SMase is responsible for ceramide generation in the CNS during normal development and particularly in response to neurotrophins [35].…”

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake

“…Similar to the protocol described previously for purification of N-SMase α, β, γ, and δ [13], a salt-extractable form of membrane-bound N-SMase ε was purified as follows. First, to prepare the enzyme source for purification of N-SMase, fresh bovine brain (5 kg) kept at −70°C was homogenized with five volumes (25 l) of homogenizing buffer V (50 mM Tris-HCl, pH 7.5; 1 mM EDTA, 3 mM MgCl 2 , 50 mM KCl, and 10 mM 2-mercaptoethanol) using a Polytron homogenizer (Model PT-MR 6000; Kinematica, Switzerland).…”

“…Several neutral SMase (N-SMase) activities have been described in mammalian brain [10], [11], [12], [13]. By using a bioinformatics-based approach, 47.5-kDa N-SMase1 [14], 71-kDa N-SMase2 [15], and 97-kDa N-SMase3 [16] have been cloned and characterized.…”

“…Previously, we reported the identification and partial purification of six forms of N-SMase from the membrane fractions of bovine brain, including four (N-SMase α, β, γ, and δ) from Triton X-100 extracts and two (N-SMase ε and ζ) from salt extracts [13]. Since N-SMase2 is already known to be extracted with Triton X-100, we focused on salt-extracted N-SMase, which may be a novel form in the membrane fractions of bovine brain.…”