Purification of neutral sphingomyelinase 2 from bovine brain and its calcium-dependent activation
- Authors
- Kim, Seok Kyun; Ahn, Kyong Hoon; Jeon, Hyung Jun; Lee, Dong Hoon; Jung, Sung Yun; Jung, Kwang Mook; Kim, Dae Kyong
- Issue Date
- Feb-2010
- Publisher
- WILEY
- Keywords
- calcium; ceramide; characterization; neutral sphingomyelinase 2; purification
- Citation
- JOURNAL OF NEUROCHEMISTRY, v.112, no.4, pp 1088 - 1097
- Pages
- 10
- Journal Title
- JOURNAL OF NEUROCHEMISTRY
- Volume
- 112
- Number
- 4
- Start Page
- 1088
- End Page
- 1097
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/22632
- DOI
- 10.1111/j.1471-4159.2009.06527.x
- ISSN
- 0022-3042
1471-4159
- 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 Ca2+-dependent and could be activated by micromolar concentrations of Ca2+ under Mg2+-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 B1, an inhibitor of the de novo ceramide synthesis pathway. The present study demonstrates the Ca2+-dependent activation of nSMase2.
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