A fluorescent plate reader assay for ceramide kinase.

Analytical Biochemistry
Anthony S Don, Hugh Rosen

Abstract

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, includin...Continue Reading

References

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Citations

Jan 27, 2009·Biochemical and Biophysical Research Communications·Anthony S Don, Hugh Rosen
Oct 4, 2008·Analytical Biochemistry·Christine GrafFrédéric Bornancin
Aug 18, 2012·Journal of Lipids·Helena Van OverloopPaul P Van Veldhoven
Dec 4, 2010·Journal of Pharmacological Sciences·Eiko TadaToshihiko Murayama
Oct 12, 2016·Biological & Pharmaceutical Bulletin·Wataru MatsuzakiToshihiko Murayama
Sep 1, 2013·Russian Journal of Bioorganic Chemistry·I A BoldyrevJ G Molotkovsky
Jul 31, 2009·Journal of Biomolecular Screening·Douglas J DemianMaura E Charlton
Sep 19, 2019·Nature Metabolism·Jong Bae SeoJerrold M Olefsky
Sep 28, 2017·Mediators of Inflammation·Marta OrdoñezAntonio Gomez-Muñoz
Nov 30, 2010·Cellular Signalling·Frédéric Bornancin

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