Purification and Characterization of Neutral and Acid Sphingomyelinases from Rat Brain

The magnesium-dependent, plasmamembrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homol-

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Magnesium-dependent Neutral Sphingomyelinase from Bovine Brain

Bernardo¹,

Krut²,

Wiegmann³

et al. 2000

“…Over the last three decades, many attempts have been made to purify this enzyme from rat brain (25,26), rat liver (27), rat ascites hepatoma (28), human brain (18,29), and human urine (30). The purification efforts so far have not been very successful, probably due to multiple factors.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Membrane Bound Neutral pH Optimum Magnesium-dependent and Phosphatidylserine-stimulated Sphingomyelinase from Rat Brain

Liu

Hassler²,

Smith³

et al. 1998

118

104

Sphingomyelin hydrolysis and ceramide generation catalyzed by sphingomyelinases (SMase) are key components of the signaling pathways in cytokine-and stressinduced cellular responses. In this study, we report the partial purification and characterization of the membrane bound, neutral pH optimal, and magnesium-dependent SMase (N-SMase) from rat brain. Proteins from Triton X-100 extract of brain membrane were purified sequentially with DEAE-Sephacel, heparin-Sepharose, ceramic hydroxyapatite, Mono Q, phenyl-Superose, and Superose 12 column chromatography. After eight purification steps, the specific activity of the enzyme increased by 3030-fold over the brain homogenate. The enzyme hydrolyzed sphingomyelin but not phosphatidylcholine and its activity was dependent upon magnesium with an optimal pH of 7.5 and a native pI of 5.2. Delipidation of the enzyme through chromatographic purification or by extraction with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid followed by gel filtration revealed that the enzyme became increasingly dependent on phosphatidylserine (PS). Up to 20-fold stimulation was observed with PS whereas other lipids examined were either ineffective or only mildly stimulatory. The K m of the enzyme for substrate sphingomyelin (3.4 mol %) was not affected by PS. The highly purified enzyme was inhibited by glutathione with a >95% inhibition observed with 3 mM glutathione and with a Hill number calculated at approximately 8. The significance of these results to the regulation of N-SMase is discussed.

“…This enzyme, which is defective in types A and B Niemann-Pick disease (11), has been purified and cloned (12), and mice in which the gene encoding lysosomal SMase has been inactivated have recently been generated (13, 14); (b) a neutral, membrane-associated, Mg 2ϩ -stimulated SMase that is found predominantly in brain and kidney (15) and that is known to arise from a separate gene from lysosomal SMase (16) Although the molecular identity and functions are definitively known only for lysosomal SMase (15), two of the other forms, namely, Mg 2ϩ -stimulated SMase and cytosolic SMase, also have been studied. Both have been partially purified (17,19,20), and these two enzymes, in addition to lysosomal SMase, have been implicated in ceramide-mediated signal transduction (17,21,22). The cellular sources, genetic origin, and functions of Zn-SMase, however, have remained totally unknown since the first and only report on this enzyme by Spence et al in 1989 (18).…”

Section: Mammalian Smasesmentioning

confidence: 99%

“…Both have been partially purified (17,19,20), and these two enzymes, in addition to lysosomal SMase, have been implicated in ceramide-mediated signal transduction (17,21,22). The cellular sources, genetic origin, and functions of Zn-SMase, however, have remained totally unknown since the first and only report on this enzyme by Spence et al in 1989 (18).…”

Section: Mammalian Smasesmentioning

confidence: 99%

Zn2+-stimulated Sphingomyelinase Is Secreted by Many Cell Types and Is a Product of the Acid Sphingomyelinase Gene

Schissel

Schuchman

Williams

et al. 1996

267

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. Although several mammalian sphingomyelinases have been identified and studied, one of these, an acidic Zn 2؉ -stimulated sphingomyelinase (Zn-SMase) originally found in fetal bovine serum, has received little attention since its first and only report 7 years ago. We now show that Zn-SMase activity is secreted by human and murine macrophages, human skin fibroblasts, microglial cells, and several other cells in culture and is markedly up-regulated during differentiation of human monocytes to macrophages. Remarkably, peritoneal macrophages from mice in which the acid SMase gene had been disrupted by homologous recombination secreted no Zn-SMase activity, indicating that this enzyme and the intracellular lysosomal SMase, which is Zn-independent, arise from the same gene. Furthermore, skin fibroblasts from patients with types A and B NiemannPick disease, which are known to lack lysosomal SMase activity, also lack Zn-SMase activity in their conditioned media. Chinese hamster ovary cells stably transfected with a cDNA encoding lysosomal SMase massively overexpress both cellular lysosomal SMase and secreted ZnSMase activities. Thus, Zn-SMase arises independently of alternative splicing, suggesting a post-translational process. In summary, a wide variety of cell types secrete Zn-SMase activity, which arises from the same gene as lysosomal SMase. This secreted enzyme may play roles in physiological and pathophysiological processes involving extracellular sphingomyelin hydrolysis. Mammalian SMases1 have been implicated in many important physiological and pathophysiological processes, including lysosomal digestion of sphingomyelin, which is important for normal neuronal and vascular function (1); ceramide-mediated signal transduction, leading to cytokine-induced apoptosis, cellular differentiation, and various immune and inflammatory responses (2, 3); lipoprotein aggregation within the vessel wall, which is a key event in atherogenesis (4 -7); and intracellular cholesterol trafficking and metabolism (8 -10).2 Several different forms of mammalian SMases have been identified, including: (a) a lysosomal SMase that is present in all tissues, acts optimally at low pH, and shows no dependence on divalent cations (11). This enzyme, which is defective in types A and B Niemann-Pick disease (11), has been purified and cloned (12), and mice in which the gene encoding lysosomal SMase has been inactivated have recently been generated (13, 14); (b) a neutral, membrane-associated, Mg 2ϩ -stimulated SMase that is found predominantly in brain and kidney (15) and that is known to arise from a separate gene from lysosomal SMase (16) Although the molecular identity and functions are definitively known only for lysosomal SMase (15), two of the other forms, namely, Mg 2ϩ -stimulated SMase and cytosolic SMase, also have been studied. Both have been partially purified (17,19,20), and these two enzymes, in addition to lysosomal SMase, ...