Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.
The magnesium-dependent, plasma membrane-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 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) 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 microM 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.
Bernardo
K
K
Institute of Medical Microbiology and Hygiene, Medical Center, University of Cologne, 50935 Köln, Germany.
Krut
O
O
Wiegmann
K
K
Kreder
D
D
Micheli
M
M
Schäfer
R
R
Sickman
A
A
Schmidt
W E
WE
Schröder
J M
JM
Meyer
H E
HE
Sandhoff
K
K
Krönke
M
M
D000577:Amides / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002412:Cations; D002417:Cattle; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology*; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D011753:Pyrones / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism
D000577:Amides; D002412:Cations; D007527:Isoenzymes; D008565:Membrane Proteins; D011753:Pyrones; C121123:scyphostatin; D013108:Sphingomyelin Phosphodiesterase; D005978:Glutathione; D008274:Magnesium