Title: A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C(6)-Ceramide
Authors: Van Overloop, Helena
Van der Hoeven, Gerd
Van Veldhoven, Paul P # ×
Issue Date: 2012
Publisher: Hindawi Publishing Corporation
Series Title: Journal of Lipids vol:2012 issue:2012 pages:404513
Article number: doi:10.1155/2012/404513
Abstract: Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-(32)P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C(6)-ceramide) as substrate. Its K(m) value (4 μM) was comparable to that of N-hexanoyl-sphingenine (C(6)-ceramide). The produced fluorescent NBD-C(6)-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC(50) 4 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC(50) 1.1 μM) affect CERK in vitro.
ISSN: 2090-3049
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Laboratory of Lipid Biochemistry and Protein Interactions
Pharmacology Section (-)
Laboratory of Biosignaling & Therapeutics
× corresponding author
# (joint) last author

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