title: A Sensitive and Versatile Fluorescent Activity Assay for ABHD6.
abstract: The α/β-hydrolase domain-containing 6 (ABHD6) enzyme is a newly found serine hydrolase whose substrate profile resembles that of monoacylglycerol lipase (MAGL), the major 2-arachidonoyl glycerol (2-AG) hydrolase in the brain. Here, we describe a sensitive fluorescent assay of ABHD6 activity in a 96-well-plate format that allows parallel testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD6 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred arachidonoyl glycerol isomer. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. The approach has major benefits compared to laborious traditional mass spectrometric methods and liquid scintillation-based assays, or approaches using unnatural substrates.
authors.affiliation: School of Medicine, Institute of Biomedicine/Physiology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Juha R
authors.initials: JR
authors.lastname: Savinainen
authors.affiliation: School of Medicine, Institute of Biomedicine/Physiology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Dina
authors.initials: D
authors.lastname: Navia-Paldanius
authors.affiliation: School of Medicine, Institute of Biomedicine/Physiology, University of Eastern Finland, Kuopio, Finland. jarmo.laitinen@uef.fi.
authors.forename: Jarmo T
authors.initials: JT
authors.lastname: Laitinen
mesh_terms: D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008994:Monoacylglycerol Lipases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; C540878:ABHD6 protein, human; D008994:Monoacylglycerol Lipases
keywords: 2-AG hydrolase; Endocannabinoid; Fluorescence; Glycerol; Inhibitor; Lipase; Monoacylglycerol; Natural substrate; Screening