Details

Autor(en) / Beteiligte

• Van Overloop, Helena
• Van der Hoeven, Gerd
• Van Veldhoven, Paul P.

Titel

A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C_6-Ceramide

Ist Teil von

• Journal of Lipids, 2012-01, Vol.2012, p.1-9

Ort / Verlag

Hindawi Limiteds

Erscheinungsjahr

2012

Link zum Volltext

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• 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  μ 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; IC 50 4  μ M) and ceramide analogues (e.g., fenretinide, AMG-9810; IC 50 1.1  μ M) affect CERK in vitro .