| Chapter title |
A Sensitive and Versatile Fluorescent Activity Assay for ABHD6
|
|---|---|
| Chapter number | 18 |
| Book title |
Endocannabinoid Signaling
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3539-0_18 |
| 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 |
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. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 5 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 2 | 40% |
| Researcher | 1 | 20% |
| Other | 1 | 20% |
| Student > Postgraduate | 1 | 20% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 3 | 60% |
| Neuroscience | 1 | 20% |
| Chemistry | 1 | 20% |