Purification of neutral sphingomyelinase 2 from bovine brain and its calcium-dependent activation.
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 B(1), an inhibitor of the de novo ceramide synthesis pathway. The present study demonstrates the Ca2+-dependent activation of nSMase2.
Kim
Seok Kyun
SK
Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-ku, Seoul, South Korea.
Ahn
Kyong Hoon
KH
Jeon
Hyung Jun
HJ
Lee
Dong Hoon
DH
Jung
Sung Yun
SY
Jung
Kwang Mook
KM
Kim
Dae Kyong
DK
D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry / Q000187:drug effects; D019556:COS Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002518:Ceramides / Q000378:metabolism; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification* / Q000378:metabolism*; D013501:Surface-Active Agents / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D000814:Aniline Compounds; D001597:Benzylidene Compounds; D002518:Ceramides; C468773:GW 4869; D007476:Ionophores; D013501:Surface-Active Agents; D014316:Tritium; D019308:Palmitic Acid; D000001:Calcimycin; D017830:Octoxynol; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; D002118:Calcium