| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 51, 34472-34479, December 18, 1998
,
, and
From the Sphingomyelin hydrolysis and ceramide generation
catalyzed by sphingomyelinases (SMase) are key components of the
signaling pathways in cytokine- and stress-induced 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 Km 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.
Departments of Medicine and Cell Biology,
Duke University Medical Center, Durham, North Carolina 27710 and the
¶ Department of Molecular Biochemistry and Molecular
Sciences, GlaxoWellcome,
Research Triangle Park, North Carolina 27709
This article has been cited by other articles:
|
|
 |
|
  M. Straczkowski, I. Kowalska, A. Nikolajuk, S. Dzienis-Straczkowska, I. Kinalska, M. Baranowski, M. Zendzian-Piotrowska, Z. Brzezinska, and J. Gorski Relationship Between Insulin Sensitivity and Sphingomyelin Signaling Pathway in Human Skeletal Muscle Diabetes, May 1, 2004; 53(5): 1215 - 1221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. V. de Avalos, Y. Okamoto, and Y. A. Hannun Activation and Localization of Inositol Phosphosphingolipid Phospholipase C, Isc1p, to the Mitochondria during Growth of Saccharomyces cerevisiae J. Biol. Chem., March 19, 2004; 279(12): 11537 - 11545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-T. Lee, J. Xu, J.-M. Lee, G. Ku, X. Han, D.-I Yang, S. Chen, and C. Y. Hsu Amyloid-{beta} peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway J. Cell Biol., January 5, 2004; 164(1): 123 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Marchesini, C. Luberto, and Y. A. Hannun Biochemical Properties of Mammalian Neutral Sphingomyelinase2 and Its Role in Sphingolipid Metabolism J. Biol. Chem., April 11, 2003; 278(16): 13775 - 13783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Czarny, J. Liu, P. Oh, and J. E. Schnitzer Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide J. Biol. Chem., February 7, 2003; 278(7): 4424 - 4430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Luberto, D. F. Hassler, P. Signorelli, Y. Okamoto, H. Sawai, E. Boros, D. J. Hazen-Martin, L. M. Obeid, Y. A. Hannun, and G. K. Smith Inhibition of Tumor Necrosis Factor-induced Cell Death in MCF7 by a Novel Inhibitor of Neutral Sphingomyelinase J. Biol. Chem., October 18, 2002; 277(43): 41128 - 41139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. BIRBES, S. EL BAWAB, Y. A. HANNUN, and L. M. OBEID Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis FASEB J, December 1, 2001; 15(14): 2669 - 2679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Tepper, P. Ruurs, T. Wiedmer, P. J. Sims, J. Borst, and W. J. van Blitterswijk Sphingomyelin Hydrolysis to Ceramide during the Execution Phase of Apoptosis Results from Phospholipid Scrambling and Alters Cell-surface Morphology J. Cell Biol., July 11, 2000; 150(1): 155 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hofmann, S. Tomiuk, G. Wolff, and W. Stoffel Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase PNAS, May 23, 2000; 97(11): 5895 - 5900. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Bernardo, O. Krut, K. Wiegmann, D. Kreder, M. Micheli, R. Schafer, A. Sickman, W. E. Schmidt, J. M. Schroder, H. E. Meyer, et al. Purification and Characterization of a Magnesium-dependent Neutral Sphingomyelinase from Bovine Brain J. Biol. Chem., March 10, 2000; 275(11): 7641 - 7647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tomiuk, M. Zumbansen, and W. Stoffel Characterization and Subcellular Localization of Murine and Human Magnesium-dependent Neutral Sphingomyelinase J. Biol. Chem., February 25, 2000; 275(8): 5710 - 5717. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. M. Hernandez, D. J. Discher, N. H. Bishopric, and K. A. Webster Rapid Activation of Neutral Sphingomyelinase by Hypoxia-Reoxygenation of Cardiac Myocytes Circ. Res., February 4, 2000; 86(2): 198 - 204. [Abstract] [Full Text]
|
|
|
|
|
|
A. C. Fensome, F. Rodrigues-Lima, M. Josephs, H. F. Paterson, and M. Katan A Neutral Magnesium-dependent Sphingomyelinase Isoform Associated with Intracellular Membranes and Reversibly Inhibited by Reactive Oxygen Species J. Biol. Chem., January 14, 2000; 275(2): 1128 - 1136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sawai, N. Domae, N. Nagan, and Y. A. Hannun Function of the Cloned Putative Neutral Sphingomyelinase as Lyso-platelet Activating Factor-Phospholipase C J. Biol. Chem., December 31, 1999; 274(53): 38131 - 38139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Marathe, G. Kuriakose, K. J. Williams, and I. Tabas Sphingomyelinase, an Enzyme Implicated in Atherogenesis, Is Present in Atherosclerotic Lesions and Binds to Specific Components of the Subendothelial Extracellular Matrix Arterioscler. Thromb. Vasc. Biol., November 1, 1999; 19(11): 2648 - 2658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Brann, R. Scott, Y. Neuberger, D. Abulafia, S. Boldin, M. Fainzilber, and A. H. Futerman Ceramide Signaling Downstream of the p75 Neurotrophin Receptor Mediates the Effects of Nerve Growth Factor on Outgrowth of Cultured Hippocampal Neurons J. Neurosci., October 1, 1999; 19(19): 8199 - 8206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takeda, M. Tashima, A. Takahashi, T. Uchiyama, and T. Okazaki Ceramide Generation in Nitric Oxide-induced Apoptosis. ACTIVATION OF MAGNESIUM-DEPENDENT NEUTRAL SPHINGOMYELINASE VIA CASPASE-3 J. Biol. Chem., April 9, 1999; 274(15): 10654 - 10660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sawai, Y. Okamoto, C. Luberto, C. Mao, A. Bielawska, N. Domae, and Y. A. Hannun Identification of ISC1 (YER019w) as Inositol Phosphosphingolipid Phospholipase C in Saccharomyces cerevisiae J. Biol. Chem., December 8, 2000; 275(50): 39793 - 39798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-J. Kroesen, B. Pettus, C. Luberto, M. Busman, H. Sietsma, L. de Leij, and Y. A. Hannun Induction of Apoptosis through B-cell Receptor Cross-linking Occurs via de Novo Generated C16-Ceramide and Involves Mitochondria J. Biol. Chem., April 20, 2001; 276(17): 13606 - 13614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. P. de Chaves, M. Bussiere, B. MacInnis, D. E. Vance, R. B. Campenot, and J. E. Vance Ceramide Inhibits Axonal Growth and Nerve Growth Factor Uptake without Compromising the Viability of Sympathetic Neurons J. Biol. Chem., September 21, 2001; 276(39): 36207 - 36214. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
