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Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

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A NOVEL ASSAY OF ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY UTILIZING FLUORESCENT SUBSTRATE(1).

Matthew C Sanderson¹, Mark E Venable¹

¹Department of Biology, Appalachian State University, 572 Rivers Street, Boone, NC 28608-2027, USA.

Journal of Phycology

|March 25, 2016

Summary

Researchers developed a novel assay to measure acyl-CoA:diacylglycerol acyltransferase (DGAT) activity using a fluorescent substrate. This sensitive and reliable method aids research in biodiesel, crops, and human diseases.

Keywords:

DGAT biofuel hyperlipidemia lipids oil triacylglycerol triglyceride

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Last Updated: Mar 23, 2026

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

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Published on: May 13, 2020

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A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Published on: October 13, 2019

Area of Science:

Biochemistry
Enzymology
Molecular Biology

Background:

Traditional assays for acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymatic activity often involve radiolabeled substrates or product assays, which can be cumbersome and pose safety concerns.
There is a need for a more direct, sensitive, and reliable method to quantify DGAT activity for diverse research applications.

Purpose of the Study:

To develop and validate a novel, direct assay for quantifying endogenous DGAT enzymatic activity.
To utilize a fluorescently labeled substrate for improved detection and quantification.

Main Methods:

The assay employs a fluorescently labeled diacylglycerol substrate, NBD-DAG (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-diacylglycerol), and oleoyl-CoA.
Microsomal protein preparations were used as the enzyme source.
DGAT activity was measured in samples from three species of algae and rat liver.

Main Results:

The novel fluorescent assay successfully quantified DGAT activity in both algal and rat liver microsomal preparations.
The developed protocol demonstrated high sensitivity and reliability in measuring enzymatic activity.
The assay provides a direct measure of endogenous DGAT activity, bypassing the need for radiolabeled compounds.

Conclusions:

This novel fluorescent assay offers a sensitive, reliable, and direct method for measuring DGAT enzymatic activity.
The assay has significant potential to advance research in areas such as biodiesel production, oilseed crop improvement, and understanding triacylglycerol-related human pathologies.