Journal
JOURNAL OF LIPID RESEARCH
Volume 53, Issue 8, Pages 1701-1707Publisher
ELSEVIER
DOI: 10.1194/jlr.D025627
Keywords
ceramides; enzymology/enzyme mechanisms; fatty acid/transferase; mass spectrometry; sphingolipids
Categories
Funding
- Cancer Institute NSW Fellowship [ECF/1-03]
- Cure Cancer Australia
- Cancer Council NSW
- NHMRC [APP1024966]
- Australian Postgraduate Award
- Cancer Therapeutics CRC PhD Top-up Scholarship
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The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, generally sphingosine or dihydrosphingosine (sphinganine) in mammals. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6). CerS activity is usually assayed using either radioactive substrates or LC-MS/MS. We describe a CerS assay with fluorescent, NBD-labeled sphinganine as substrate. The assay is readily able to detect endogenous CerS activity when using amounts of cell or tissue homogenate protein that are lower than those reported for the radioactive assay, and the Michaelis-Menten constant was essentially the same for NBD-sphinganine and unlabeled sphinganine, indicating that NBD-sphinganine is a good substrate for these enzymes. Using our assay, we confirm that the new clinical immunosuppressant FTY720 is a competitive inhibitor of CerS activity, and show that inhibition requires the compound's lipid tail and amine head-group. In summary, we describe a fluorescent assay for CerS activity that circumvents the need to use radioactive substrates, while being more accessible and cheaper than LC-MS based assays.-Kim, H. J., Q. Qiao, H. D. Toop, J. C. Morris, and A. S. Don. A fluorescent assay for ceramide synthase activity. J. Lipid Res. 2012. 53: 1701-1707.
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