Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L

Methods Mol Biol. 2017:1557:207-212. doi: 10.1007/978-1-4939-6780-3_19.

Abstract

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.

Keywords: Apoptosis; Ceramide; In situ sphingomyelin synthase activity; Sphingomyelin.

MeSH terms

  • Animals
  • Apoptosis
  • Cell Line
  • Chromatography, Thin Layer
  • Enzyme Activation
  • Enzyme Assays / methods*
  • Fas Ligand Protein / metabolism*
  • Mice
  • Protein Binding
  • Sphingomyelins / isolation & purification
  • Sphingomyelins / metabolism
  • Transferases (Other Substituted Phosphate Groups) / metabolism*

Substances

  • Fas Ligand Protein
  • Sphingomyelins
  • Transferases (Other Substituted Phosphate Groups)
  • phosphatidylcholine-ceramide phosphocholine transferase