Spotlight: Ceramide Synthase (CerS) Assay Kit
February 26, 2016
Ceramide synthases (CerSs) acylate sphingoid long-chain bases to form (dihydro)ceramides . Mammals contain six distinct CerSs, each of which uses a specific subset of fatty acyl-coenzyme As to produce various subspecies of (dihydro)ceramides . CerSs have become of considerable interest due to the growing involvement of this enzyme family in cell regulation and the pathology of human disease . In a recent publication by Tidhar, et al. in the Journal of Lipid Research, a rapid and reliable method for assaying CerS activity using only small amounts of biological material is described . This assay uses NBD sphinganine and a fatty acyl coenzyme A as the substrates for the enzyme . Upon acylation the lipid product, NBD (dihydro)ceramide, is separated from the reaction mixture using SPE column chromatography in a convenient 96-well plate format, and the formation of the fluorometric product is monitored using a standard multiwell plate reader . The 96-well SPE column chromatography format allows multiple assays to be run simultaneously . This assay is now available as a kit from Avanti. The CerS Assay Kit is sufficient for ~275 reactions and includes NBD sphinganine and one of the following fatty acyl coAs: 18:0 (CerS1/4), 16:0 (CerS5/6), or 24:1 (CerS2). The CerS Assay Kit offers a number of advantages including: a short assay time, a requirement of only small amounts of biological material, and elimination of the use of TLC as a separation technique, which has been well documented to result in the degradation of NBD sphinganine . This collaborative work with Tidhar, et al. provides a simple and high-throughput assay kit for measuring CerS activity in biological samples and provides an accessible method of analysis for laboratories that do not routinely assay enzymes of lipid metabolism .
- Tidhar, R., Sims, K., Rosenfeld-Gur, E., Shaw, W., Futerman, A. A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction. Journal of Lipid Research, 2015, 56(1):193-9.