title: Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays.
abstract: 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.
authors.affiliation: LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Gaebler
authors.affiliation: LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Robin
authors.initials: R
authors.lastname: Milan
authors.affiliation: LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Leon
authors.initials: L
authors.lastname: Straub
authors.affiliation: LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Hoelper
authors.affiliation: LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Kuerschner
authors.affiliation: LIMES Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, Germany. Electronic address: cthiele@uni-bonn.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Thiele
mesh_terms: D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000032:analysis / Q000378:metabolism*; D000217:Acyltransferases / Q000032:analysis / Q000378:metabolism*; D000480:Alkynes / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D057930:Click Chemistry; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D010088:Oxidoreductases / Q000032:analysis / Q000172:deficiency / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000480:Alkynes; D008055:Lipids; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D000217:Acyltransferases; D008243:1-Acylglycerophosphocholine O-Acyltransferase; C018764:2-acylglycerophosphate acyltransferase
keywords: acyltransferase; ceramide synthase; click lipid; click-labeling; enzyme kinetics; fatty acid amide hydrolase; in vitro assay; lipid substrate