| Autor: |
Bernardo, Katussevani, Krut, Oleg, Wiegmann, Katja, Kreder, Dirk, Micheli, Marta, Schäfer, Reiner, Sickman, Albert, Schmidt, Wolfgang E., Schröder, Jens M., Meyer, Helmut E., Sandhoff, Konrad, Krönke, Martin |
| Zdroj: |
Journal of Biological Chemistry; March 2000, Vol. 275 Issue: 11 p7641-7647, 7p |
| Abstrakt: |
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 Kmof 40 μmfor 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 μmscyphostatin. 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. |
| Databáze: |
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