文章基本信息

标题：A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction
本地全文：下载
作者：Rotem Tidhar ; Kacee Sims ; Eden Rosenfeld-Gur 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2015
卷号：56
期号：1
页码：193-199
DOI：10.1194/jlr.D052001
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N -acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.
关键词：c eramides ; sphingolipids ; in vitro assay ; chromatography ; enzymology ; long-chain base ; fatty acyl-CoA ; N -acylation