LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
J Lipid Res. 2013 Aug;54(8):2282-2290. doi: 10.1194/jlr.D038653. Epub 2013 May 23.
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.
点击化学正在发展成为生物应用中的一种强大工具，因为它允许对具有炔基或叠氮基团的化合物进行灵敏和特异性检测。在这里，我们描述了炔烃脂质作为体外酶促测定脂质修饰酶的底物的用途。小炔基部分是在各种底物脂质的烃链末端通过合成引入的。测定后，用带有叠氮基的荧光染料 3-叠氮-7-羟基香豆素点击反应标记，然后通过薄层层析和荧光检测分离脂质混合物，从而实现高灵敏度和宽线性范围。使用炔烃标记的底物进行溶血磷脂酸酰基转移酶、溶血磷脂酰胆碱酰基转移酶和神经酰胺合酶的动力学分析得到的米氏常数与放射性标记或天然底物相似。我们在脂质代谢中对几种水解酶和酰基转移酶进行了其他炔烃底物的测试。在这项初步研究中，我们将炔烃脂质确立为体外酶促测定的一类新的方便底物。
