文章基本信息

标题：A novel assay for the introduction of the vinyl ether double bond into plasmalogens using pyrene-labeled substrates
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作者：Ernst R. Werner ; Markus A. Keller ; Sabrina Sailer 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：2018
卷号：59
期号：5
页码：901-909
DOI：10.1194/jlr.D080283
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1- O -pyrenedecyl-2-acyl- sn -glycero-3-phosphoethanolamine, and the lyso-substrate, 1- O -pyrenedecyl- sn -glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1- O -pyrenedecyl- sn -glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.
关键词：ether lipid ; plasmanylethanolamine desaturase ; phosphatidyl ethanolamine ; lyso-phosphatidyl ethanolamine