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J Biol Chem, Vol. 275, Issue 11, 7641-7647, March 17, 2000

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

Katussevani Bernardo, Oleg Krut, Katja Wiegmann, Dirk Kreder^§, Marta Micheli, Reiner Schäfer^¶, Albert Sickman, Wolfgang E. Schmidt^¶, Jens M. Schröder^**, Helmut E. Meyer, Konrad Sandhoff, and Martin Krönke^§§

From the  Institute of Medical Microbiology and Hygiene, Medical Center, University of Cologne, 50935 Köln, Germany,  Institute of Physiological Chemistry, University of Bochum, 44780 Bochum, Germany, ^¶ Department of Internal Medicine and ^** Department of Dermatology, Medical Center, University of Kiel, 24105 Kiel, Germany,  Kekule'-Institute for Organic Chemistry and Biochemistry, University of Bonn, 53121 Bonn, Germany

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 homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K_m of 40 µM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg²⁺, whereas Zn²⁺ and Ca²⁺ proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 µM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

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^* This work was supported by grants from the Deutsche Forschungsgemeinschaft kr810/12-114.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Roche Bioscience, Palo Alto, CA 94304.

^§§ To whom correspondence should be addressed: Inst. of Medical Microbiology and Hygiene, Medical Center, University of Cologne, Goldenfels Strasse 19-21, 50935 Köln, Germany. Tel.: (49)221-478-3060; Fax: (49)221-478-3067; E-mail: Martin.Kroenke@medizin.uni-koeln.de.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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