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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)

Abstract

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 

Abbreviations

CERS

Ceramide

HPLC

High-performance liquid chromatography

NBD-dhCer

NBD-dihydroceramide

NBD-dhSph

NBD-dihydrosphingosine

TLC

Thin-layer chromatography

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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

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