A fluorescent assay for ceramide synthase activity.
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.
Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine,  University of New South Wales, Sydney, 2052, NSW,  Australia.
Hyun Joon
HJ
Kim
Qiao
Q
Qiao
Hamish D
HD
Toop
Jonathan C
JC
Morris
Anthony S
AS
Don
D057075:Enzyme Assays / Q000379:methods*; D005240:Feasibility Studies; D000068876:Fingolimod Hydrochloride; D005456:Fluorescent Dyes / Q000737:chemistry; D006018:Glycols / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D057230:Limit of Detection; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011409:Propylene Glycols / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D005456:Fluorescent Dyes; D006018:Glycols; D011409:Propylene Glycols; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; C005682:safingol