Title : Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta - Singh_2016_Biochemistry_55_2713 |
Author(s) : Singh PK , Markwick R , Lu L , Howell FV , Williams G , Doherty P |
Ref : Biochemistry , 55 :2713 , 2016 |
Abstract :
The diacylglycerol lipases (DAGLalpha and DAGLbeta) hydrolyze DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions toward 2-AG and AA levels, and therefore, selective modulators for these enzymes could play crucial roles toward harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLalpha and have proven useful toward the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLbeta which was the aim of this study. We first adapted previously reported DAGLalpha membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLbeta activity in membranes. In contrast to results with DAGLalpha, both substrates provided a relatively limited signal window for measuring DAGLbeta activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLbeta. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors (THL and OMDM-188). The purified DAGLbeta catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts. |
PubMedSearch : Singh_2016_Biochemistry_55_2713 |
PubMedID: 27115711 |
Gene_locus related to this paper: human-DAGLB |
Inhibitor | L-Isoleucine-Orlistat |
Gene_locus | human-DAGLB |
Singh PK, Markwick R, Lu L, Howell FV, Williams G, Doherty P (2016)
Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta
Biochemistry
55 :2713
Singh PK, Markwick R, Lu L, Howell FV, Williams G, Doherty P (2016)
Biochemistry
55 :2713