Fluorescence-Based NAPE-PLD Activity Assay

Elliot D Mock; Wouter P F Driever; Mario van der Stelt

doi:10.1007/978-1-0716-2728-0_19

Fluorescence-Based NAPE-PLD Activity Assay

Methods Mol Biol. 2023:2576:233-240. doi: 10.1007/978-1-0716-2728-0_19.

Authors

Elliot D Mock¹, Wouter P F Driever², Mario van der Stelt²

Affiliations

¹ Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University & Oncode Institute, RA, Leiden, The Netherlands. elliot.mock@dpag.ox.ac.uk.
² Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University & Oncode Institute, RA, Leiden, The Netherlands.

PMID: 36152191
DOI: 10.1007/978-1-0716-2728-0_19

Abstract

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the principal enzyme that generates N-acylethanolamines (NAEs), a family of signaling lipids that includes the endocannabinoid anandamide. To investigate the biological function and biosynthesis of NAEs, we sought to develop potent NAPE-PLD inhibitors. To this aim, we utilized a high-throughput screening-compatible NAPE-PLD activity assay, which uses the fluorescence-quenched substrate PED6. This assay conveniently uses membrane fractions of NAPE-PLD overexpressing HEK293T cell lysates, thus avoiding the need for protein purification. Here, we give a detailed description of the NAPE-PLD PED6 fluorescence activity assay, which has increased throughput compared to previous radioactivity- or mass-spectrometry-based assays.

Keywords: Anandamide; Fluorescence-based activity assay; Inhibitor screening; N-Acylethanolamines; N-Acylphosphatidylethanolamine phospholipase D; NAPE-PLD; PED6.

MeSH terms

Endocannabinoids*
Fluorescence
HEK293 Cells
Humans
Phospholipase D* / metabolism

Substances

Endocannabinoids
Phospholipase D