A fluorescent plate reader assay for ceramide kinase.
Ceramide kinase and its product ceramide 1-phosphate have been implicated in cellular proliferation and survival, activation of cytosolic phospholipase A(2), mast cell degranulation, and phagocytosis. Current assays for ceramide kinase activity employ [(32)P]ATP, with separation of labeled product from excess ATP by organic extraction and thin-layer chromatography. We have developed a fluorescent plate reader assay for ceramide kinase that uses commercially available C6-NBD ceramide (N-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine). Our assay is based on the differential partitioning of substrate and product following a single chloroform/methanol extraction. The product, which partitions into the aqueous phase at physiological pH, is quantitated directly in a plate reader. The substrate may be delivered using either fatty acid-free albumin or detergent/lipid mixed micelles, and we have found that the use of albumin rather than detergent micelles allows one to detect lipid interactions with the enzyme that might otherwise go unnoticed. Our method is useful for assaying ceramide kinase activity both in vitro and in cultured cells, and it offers several advantages over the conventional assay, including greater speed, the ability to run a larger number of assay replicates at one time, and the elimination of environmental and safety issues associated with the use of radioactive materials.
Don
Anthony S
AS
Department of Chemical Physiology, Scripps Research Institute, La Jolla, CA 92037, USA.
Rosen
Hugh
H
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation*; D002460:Cell Line; D002518:Ceramides / Q000302:isolation & purification / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism
D002518:Ceramides; C065576:ceramide 1-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D013110:Sphingosine