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A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction.
- Source :
-
Journal of lipid research [J Lipid Res] 2015 Jan; Vol. 56 (1), pp. 193-9. Date of Electronic Publication: 2014 Nov 03. - Publication Year :
- 2015
-
Abstract
- Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.<br /> (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- 4-Chloro-7-nitrobenzofurazan isolation & purification
4-Chloro-7-nitrobenzofurazan metabolism
HEK293 Cells
Humans
Time Factors
4-Chloro-7-nitrobenzofurazan analogs & derivatives
Ceramides isolation & purification
Ceramides metabolism
Enzyme Assays methods
Oxidoreductases metabolism
Solid Phase Extraction
Subjects
Details
- Language :
- English
- ISSN :
- 1539-7262
- Volume :
- 56
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of lipid research
- Publication Type :
- Academic Journal
- Accession number :
- 25368106
- Full Text :
- https://doi.org/10.1194/jlr.D052001