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Related Experiment Video

Updated: Aug 27, 2025

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening
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Fluorescence-Based NAPE-PLD Activity Assay.

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

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

Methods in Molecular Biology (Clifton, N.J.)
|September 24, 2022
PubMed
Summary
This summary is machine-generated.

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N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) generates signaling lipids. A new fluorescence assay enables high-throughput screening for NAPE-PLD inhibitors, aiding NAE research.

Area of Science:

  • Biochemistry
  • Lipid signaling
  • Enzyme kinetics

Background:

  • N-acylethanolamines (NAEs) are crucial signaling lipids, including anandamide.
  • N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is the primary enzyme responsible for NAE biosynthesis.
  • Understanding NAE function and production requires tools to study NAPE-PLD activity.

Purpose of the Study:

  • To develop potent inhibitors of NAPE-PLD.
  • To establish a high-throughput screening (HTS) compatible assay for NAPE-PLD activity.
  • To facilitate the investigation of NAE biological functions and biosynthesis.

Main Methods:

  • Development of a fluorescence-based NAPE-PLD activity assay using the substrate PED6.
  • Utilized membrane fractions from NAPE-PLD overexpressing HEK293T cell lysates.
Keywords:
AnandamideFluorescence-based activity assayInhibitor screeningN-AcylethanolaminesN-Acylphosphatidylethanolamine phospholipase DNAPE-PLDPED6

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  • Compared throughput with existing radioactivity- and mass-spectrometry-based assays.

    • Main Results:

    • A detailed protocol for the NAPE-PLD PED6 fluorescence activity assay is described.
    • The developed assay is compatible with high-throughput screening.
    • This method avoids the need for protein purification, simplifying the process.

    Conclusions:

    • The NAPE-PLD PED6 fluorescence assay offers a high-throughput alternative for studying NAPE-PLD.
    • This assay facilitates the discovery of NAPE-PLD inhibitors.
    • Enables further research into the roles of NAEs in biological systems.