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KMID : 0578320230460090545
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Molecules and Cells
2023 Volume.46 No. 9 p.545 ~ p.557
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Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain
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Choi Jong-Min
Yongwei Piao
Ahn Kyong-Hoon
Kim Seok-Kyun
Won Jong-Hoon
Lee Jae-Hong
Jang Ji-Min
Shin In-Chul
Zhicheng Fu
Jung Sung-Yun
Jeong Eui-Man
Kim Dae-Kyong
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Abstract
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Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, N2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.
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KEYWORD
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brain, Mg2+-independent sphingomyelinase, mitochondria, purification
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