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Updated: Jul 10, 2026
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Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
Biochemical analysis of phospholipase D.
H Alex Brown1, Lee G Henage, Anita M Preininger
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Methods in Enzymology
|October 24, 2007
Summary
Phospholipase D (PLD) is a crucial enzyme in cell signaling. This study details methods for producing and assaying recombinant PLD1, including in vitro and in vivo assays, and novel fluorescence-based assays.
Area of Science:
- Biochemistry
- Enzymology
- Cell Biology
Background:
- Phospholipase D (PLD) is a ubiquitous enzyme catalyzing phospholipid hydrolysis.
- PLD plays key roles in cell signaling through the production of phosphatidic acid (PA).
- Understanding PLD activity requires robust and versatile assay methods.
Purpose of the Study:
- To describe the production and purification of recombinant mammalian PLD1.
- To detail various in vitro and in vivo assay methods for PLD activity.
- To present novel fluorescence-based assays for PLD and diacylglycerol (DAG) lipase.
Main Methods:
- Recombinant mammalian PLD1 production in baculovirus-infected insect cells.
- In vitro assays measuring choline release or transphosphatidylation using radiolabeled substrates.
- In vivo assays using (3)H-labeled fatty acids and mass spectrometry with deuterated substrates.
- Development of fluorescence-based assays for PLD and DAG lipase.
Main Results:
- Successful production and purification of active recombinant PLD1.
- Established and validated diverse assay methodologies for PLD.
- Demonstrated utility of transphosphatidylation assays, including mass spectrometry-based approaches.
- Introduced convenient fluorescence-based assays for PLD and DAG lipase activity.
Conclusions:
- The described methods enable comprehensive study of PLD activity in various biological contexts.
- Novel fluorescence assays offer convenient and sensitive alternatives for enzyme activity measurement.
- These assays facilitate research into PLD function and regulation in cellular processes.
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