Singh_2016_Biochemistry_55_2713

Reference

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

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Gene_locus human-DAGLB

Citations formats

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