Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain

  • Jong Min Choi (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Yongwei Piao (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Kyong Hoon Ahn (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Seok Kyun Kim (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Jong Hoon Won (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Jae Hong Lee (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Ji Min Jang (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • In Chul Shin (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Zhicheng Fu (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Sung Yun Jung (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Eui Man Jeong (Department of Pharmacy, College of Pharmacy, Jeju Research Institute of Pharmaceutical Sciences, Jeju National University) ;
  • Dae Kyong Kim (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University)
  • Received : 2023.05.04
  • Accepted : 2023.05.16
  • Published : 2023.09.30
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Abstract

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+, Ni2+, 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.

Keywords

Acknowledgement

This research was supported by the National Research Foundation (NRF) grant funded by the Korea government (MSIT and MOE, grant Nos. NRF-2020R1A2C1102124 and 2019R1A6A1A10072987, respectively).

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