Title : A Sensitive and Versatile Fluorescent Activity Assay for ABHD6 - Savinainen_2016_Methods.Mol.Biol_1412_169 |
Author(s) : Savinainen JR , Navia-Paldanius D , Laitinen JT |
Ref : Methods Mol Biol , 1412 :169 , 2016 |
Abstract :
The alpha/beta-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. |
PubMedSearch : Savinainen_2016_Methods.Mol.Biol_1412_169 |
PubMedID: 27245903 |
Savinainen JR, Navia-Paldanius D, Laitinen JT (2016)
A Sensitive and Versatile Fluorescent Activity Assay for ABHD6
Methods Mol Biol
1412 :169
Savinainen JR, Navia-Paldanius D, Laitinen JT (2016)
Methods Mol Biol
1412 :169