Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α

A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α

“…We used the linear interval of t = 10 to t = 35 min to determine the reaction rate of the enzyme. Interestingly, and in contrast to previous reports ( 4,8 ), the addition of calcium in the assay buffer did not result in an increase of the DAGL-␣ -specifi c signal, possibly because this affected the downstream enzymatic cascade.…”

“…These values fall within the same range as those previously reported by two independent groups using a radiometric and an LC/MS-based assay ( 4,8 ). It should be emphasized that measured kinetic parameters for lipases are apparent values, which are dependent on the employed assay conditions, such as the limited solubility of the lipophilic substrates in aqueous media and the use of membrane preparations instead of purifi ed enzyme.…”

Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta

“…Possible explanations for this difference could be higher background enzymatic activity in the parental membranes towards DiFMUO when compared to PNPB which could especially be problematic as DiFMUO may access the DAGLs through the membrane whereas PNPB may access the DAGLs through the aqueous phase. If this is the case, then the membrane diffusion rates of DiFMUO could prove to be 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 rate limiting in the context of DAGL activity 33 . If both substrates were to access the DAGLs through the membrane, then different membrane diffusion rates could explain the differences in activity.…”

“…For further experiments 500ng of enzyme was used per well. We next determined the effect of substrate concentration on the rate of activity ( Figure 3C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 more potent DAGL inhibitor 33,35,36 . The intra and inter-assay coefficient of variations were determined as 4.5% (n=4) and 9.8% (N=5) respectively.…”

“…Van der Wel et al reported on a DAGLα native substrate assay using a coupled enzyme approach adapted from a previously reported MAGL assay 31,32 . Pedicord et al, reported on simple economical recombinant DAGLα membrane based assays utilizing the fluorogenic 6, 8-difluoro-4-methylumbelliferyl-octanoate (DiFMUO) and the chromogenic p-nitrophenyl butyrate (PNPB) as surrogate substrates 33,34 .…”