A Nonradioactive Fluorimetric Spe-Based Ceramide Kinase Assay Using Nbd-C-6-Ceramide

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [gamma-P-32]-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 Km value (4 mu M) 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; IC50 4 mu M) and ceramide analogues (e.g., fenretinide, AMG-9810; IC50 1.1 mu M) affect CERK in vitro.