In:
Journal of Lipids, Hindawi Limited, Vol. 2012 ( 2012), p. 1-9
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 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 .
Type of Medium:
Online Resource
ISSN:
2090-3030
,
2090-3049
Language:
English
Publisher:
Hindawi Limited
Publication Date:
2012
detail.hit.zdb_id:
2582309-7
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