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Purification of neutral sphingomyelinase 2 from bovine brain and its calcium‐dependent activation
Author(s) -
Kim Seok Kyun,
Ahn Kyong Hoon,
Jeon Hyung Jun,
Lee Dong Hoon,
Jung Sung Yun,
Jung Kwang Mook,
Kim Dae Kyong
Publication year - 2010
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2009.06527.x
Subject(s) - sphingomyelin , ceramide , ceramide synthase , calcium , biochemistry , apoptosis , chemistry , ionophore , microbiology and biotechnology , biology , membrane , organic chemistry
J. Neurochem. (2010) 112 , 1088–1097. Abstract Ceramide is produced by sphingomyelinase (SMase) and it plays a key role in cellular responses such as apoptosis. In this study, we report the purification and characterization of neutral SMase2 (nSMase2) from bovine brain tissue. Triton X‐100 extracts of bovine brain membranes were purified in nine steps, including sequential chromatography. The specific activity of purified nSMase increased 8183‐fold over the brain membrane fraction. Purified nSMase showed similarities to nSMase2, which had been purified and cloned previously. Interestingly, purified nSMase2 was Ca 2+ ‐dependent and could be activated by micromolar concentrations of Ca 2+ under Mg 2+ ‐free conditions. Ceramide generation was dependent upon the calcium ionophore A23187 and was observed in nSMase2‐over‐expressing COS‐7 cells. This generation was suppressed by GW4869, an nSMase2 inhibitor, but not to fumonisin B 1 , an inhibitor of the de novo ceramide synthesis pathway. The present study demonstrates the Ca 2+ ‐dependent activation of nSMase2.