Fluorescent Assays for Ceramide Synthase Activity

  • Timothy A. Couttas
  • Anthony S. DonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)


Ceramides are the central lipid metabolite of the sphingolipid family, and exert a potent influence over cell polarity, differentiation, and survival through their biophysical properties and their specific interactions with cell signaling proteins. Literature on the importance of ceramides in physiology and pathological conditions continues to grow, with ceramides having been identified as central effectors in major human pathologies such as diabetes and neurodegenerative conditions. In mammals, ceramide synthesis from a sphingoid base and a variable length fatty acid is catalyzed by a family of six ceramide synthases (CERS1-6), whose active sites exhibit differential specificity for different length fatty acids. CERS activity has traditionally been measured using radioactive substrates. More recently mass spectrometry has been used. In this chapter, we describe a fluorescent CERS assay, the results of which can be quantified using thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Methods for quantification with either TLC or HPLC are described.

Key words

Ceramide Ceramide synthase Fluorescent Assay 





High-performance liquid chromatography






Thin-layer chromatography


  1. 1.
    Park JW, Park WJ, Futerman AH (2014) Ceramide synthases as potential targets for therapeutic intervention in human diseases. Biochim Biophys Acta 1841(5):671–681PubMedCrossRefGoogle Scholar
  2. 2.
    Hannun YA, Obeid LM (2011) Many ceramides. J Biol Chem 286(32):27855–27862PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Chavez JA, Summers SA (2012) A ceramide-centric view of insulin resistance. Cell Metab 15(5):585–594PubMedCrossRefGoogle Scholar
  4. 4.
    Haughey NJ, Bandaru VV, Bae M, Mattson MP (2010) Roles for dysfunctional sphingolipid metabolism in Alzheimer’s disease neuropathogenesis. Biochim Biophys Acta 1801(8):878–886PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    de la Monte SM (2012) Triangulated mal-signaling in Alzheimer’s disease: roles of neurotoxic ceramides, ER stress, and insulin resistance reviewed. J Alzheimers Dis 30(Suppl 2):S231–S249PubMedPubMedCentralGoogle Scholar
  6. 6.
    Bieberich E (2008) Ceramide signaling in cancer and stem cells. Future Lipidol 3(3):273–300PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Narayanaswamy P, Shinde S, Sulc R, Kraut R, Staples G, Thiam CH, Grimm R, Sellergren B, Torta F, Wenk MR (2014) Lipidomic “deep profiling”: an enhanced workflow to reveal new molecular species of signaling lipids. Anal Chem 86(6):3043–3047PubMedCrossRefGoogle Scholar
  8. 8.
    Merrill AH Jr (2011) Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem Rev 111(10):6387–6422PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Mizutani Y, Kihara A, Igarashi Y (2005) Mammalian Lass6 and its related family members regulate synthesis of specific ceramides. Biochem J 390(Pt 1):263–271PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Jennemann R, Rabionet M, Gorgas K, Epstein S, Dalpke A, Rothermel U, Bayerle A, van der Hoeven F, Imgrund S, Kirsch J, Nickel W, Willecke K, Riezman H, Grone HJ, Sandhoff R (2012) Loss of ceramide synthase 3 causes lethal skin barrier disruption. Hum Mol Genet 21(3):586–608PubMedCrossRefGoogle Scholar
  11. 11.
    Lahiri S, Lee H, Mesicek J, Fuks Z, Haimovitz-Friedman A, Kolesnick RN, Futerman AH (2007) Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine. FEBS Lett 581(27):5289–5294PubMedCrossRefGoogle Scholar
  12. 12.
    Bose R, Kolesnick R (2000) Measurement of ceramide synthase activity. Methods Enzymol 322:378–382PubMedCrossRefGoogle Scholar
  13. 13.
    Kim HJ, Qiao Q, Toop HD, Morris JC, Don AS (2012) A fluorescent assay for ceramide synthase activity. J Lipid Res 53(8):1701–1707PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Couttas TA, Lim XY, Don AS (2015) A three-step assay for ceramide synthase activity using a fluorescent substrate and HPLC. Lipids 50:101–109PubMedCrossRefGoogle Scholar
  15. 15.
    Muir A, Ramachandran S, Roelants FM, Timmons G, Thorner J (2014) TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids. Elife 3Google Scholar
  16. 16.
    Merrill AH Jr, van Echten G, Wang E, Sandhoff K (1993) Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J Biol Chem 268(36):27299–27306PubMedGoogle Scholar
  17. 17.
    Ohno Y, Suto S, Yamanaka M, Mizutani Y, Mitsutake S, Igarashi Y, Sassa T, Kihara A (2010) ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis. Proc Natl Acad Sci U S A 107(43):18439–18444PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Sassa T, Suto S, Okayasu Y, Kihara A (2012) A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells. Biochim Biophys Acta 1821(7):1031–1037PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Prince of Wales Clinical School, Faculty of MedicineUniversity of New South WalesSydneyAustralia
  2. 2.SydneyAustralia

Personalised recommendations