Abstract
The α/β-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.
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References
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Acknowledgement
This work was supported by the Academy of Finland (Grant 139620 to J.T.L.).
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Savinainen, J.R., Navia-Paldanius, D., Laitinen, J.T. (2016). A Sensitive and Versatile Fluorescent Activity Assay for ABHD6. In: Maccarrone, M. (eds) Endocannabinoid Signaling. Methods in Molecular Biology, vol 1412. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3539-0_18
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DOI: https://doi.org/10.1007/978-1-4939-3539-0_18
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3537-6
Online ISBN: 978-1-4939-3539-0
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