文章基本信息

标题：Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays
本地全文：下载
作者：Anne Gaebler ; Robin Milan ; Leon Straub 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：2013
卷号：54
期号：8
页码：2282-2290
DOI：10.1194/jlr.D038653
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.
关键词：ceramide synthase ; acyltransferase ; fatty acid amide hydrolase ; click lipid ; click-labeling ; lipid substrate ; enzyme kinetics ; in vitro assay