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Type: Journal article
Title: A fluorescent assay for ceramide synthase activity
Author: Kim, H.J.
Qiao, Q.
Toop, H.D.
Morris, J.C.
Don, A.S.
Citation: Journal of Lipid Research, 2012; 53(8):1701-1707
Publisher: American Society for Biochemistry and Molecular Biology
Issue Date: 2012
ISSN: 0022-2275
Statement of
Hyun Joon Kim, Qiao Qiao, Hamish D. Toop, Jonathan C. Morris and Anthony S. Don
Abstract: 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 headgroup. 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.
Keywords: Ceramides; enzymology/enzyme mecha-nisms; fatty acid/transferase; mass spectrometry; sphingolipid
Rights: © 2012 by the American Society for Biochemistry and Molecular Biology, Inc. This is an Open Access article under the CC BY license.
DOI: 10.1194/jlr.D025627
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