Chapter title |
A Sensitive and Versatile Fluorescent Activity Assay for ABHD12
|
---|---|
Chapter number | 19 |
Book title |
Endocannabinoid Signaling
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3539-0_19 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3537-6, 978-1-4939-3539-0
|
Authors |
Juha R. Savinainen, Dina Navia-Paldanius, Jarmo T. Laitinen |
Editors |
Mauro Maccarrone |
Abstract |
Despite great progress in identifying and deorphanizing members of the human metabolic serine hydrolase (mSH) family, the fundamental role of numerous enzymes in this large protein class has remained unclear. One recently found mSH is α/β-hydrolase domain containing 12 (ABHD12) enzyme, whose natural substrate in vivo appears to be the lysophospholipid lysophosphatidylserine (LPS). In vitro, ABHD12 together with monoacylglycerol lipase (MAGL) and ABHD6 hydrolyzes also monoacylglycerols (MAGs) such as the primary endocannabinoid 2-arachidonoyl glycerol (2-AG). Traditional approaches for determining 2-AG hydrolase activity are rather laborious, and often utilize unnatural substrates. Here, we describe a sensitive fluorescent assay of ABHD12 activity in a 96-well-plate format that allows simultaneous testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD12 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred MAG substrate of this enzyme. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. This methodology has helped to identify the first class of inhibitors showing selectivity for ABHD12 over the other mSHs. |
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