Fluorescent Assays for Ceramide Synthase Activity
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 wordsCeramide Ceramide synthase Fluorescent Assay
High-performance liquid chromatography
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