Direct and Continuous Measurement of Phospholipase D Activities Using the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1835; p. 129
• Main Authors: Rahier, Renaud, Abla, Houda, Arhab, Yani, Noiriel, Alexandre, Abousalham, Abdelkarim
• Format: Journal Article
• Language: English
• Published: United States 01.01.2018
• Subjects: Enzyme Activation; Enzyme Assays - methods; Enzyme Assays - standards; Fluorescent Dyes; Humans; Hydrolysis; Kinetics; Oxyquinoline - metabolism; Phospholipase D - chemistry; Phospholipase D - metabolism; 8-Hydroxyquinoline; Assay; Screening; Inhibitors; Fluorescence; Microplate; Transphosphatidylation; Phospholipase D; Phosphatidic acid
• DOI: 10.1007/978-1-4939-8672-9_6

Phospholipase D (PLD) hydrolyzes phospholipids to form phosphatidic acid (PA) and the corresponding headgroup. To date, PLD has been linked to several pathologies, such as cancer, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released upon phosphatidylcholine (PC) hydrolysis. Therefore, we designed a PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline. This assay exhibits a strong fluorescence signal upon Ca complexation with the PLD-generated PA and is not limited to PC as the substrate but allows the use of natural phospholipids with various headgroups. Besides, this easy-to-handle assay allows to monitor prokaryotic and eukaryotic PLD activities in a continuous way and on a microplate scale.