A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide

Abstract EN

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-32P]-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-C6-ceramide) as substrate.

Its Km value (4 μM) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide).

The produced fluorescent NBD-C6-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 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC50 1.1 μM) affect CERK in vitro.