A three-step assay for ceramide synthase activity using a fluorescent substrate and HPLC.
Ceramides are a family of signalling lipids with diverse physiological functions that include pro-differentiative and pro-apoptotic signalling. Ceramides and their derivatives are major constituents of myelin, maintaining neuronal conductivity. Ceramides are synthesized by ceramide synthases, of which there are six isoforms in mammals (CERS1-6). These enzymes catalyse the transfer of a variable length fatty acid to a sphingoid base, typically sphingosine or dihydrosphingosine. We previously reported a fluorescent thin-layer chromatography assay for ceramide synthase activity. In this paper we describe an improved fluorescent assay, using HPLC to achieve clear resolution of closely related ceramide species and to facilitate easy quantification of both product and substrate. Our HPLC assay protocol eliminates the need for a chloroform extraction step. Instead a simple three-step procedure is used: (1) reactions are run; (2) reactions are terminated with addition of methanol and centrifuged; (3) products are quantified with HPLC. HPLC resolution enables assays in which multiple fatty acid substrates are used in the same reaction. Using this approach, we show that CERS2 demonstrates a preference for the monounsaturated C24:1 fatty acid substrate compared to the saturated C24:0 substrate, potentially explaining why myelin is enriched in ceramides containing the monounsaturated form of very long chain fatty acids.
Prince of Wales Clinical School, University of New South Wales, Level 2, C25 Lowy Building, Sydney, NSW, 2052, Australia.
Timothy A
TA
Couttas
Xin Y
XY
Lim
Anthony S
AS
Don
D045744:Cell Line, Tumor; D002518:Ceramides / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D057230:Limit of Detection; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D051049:Sphingosine N-Acyltransferase / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000378:metabolism*
D002518:Ceramides; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D025521:Tumor Suppressor Proteins; C438311:CERS2 protein, human; D051049:Sphingosine N-Acyltransferase