Chapter 6 Direct and Continuous Measurement of Phospholipase D Activities Using the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline Renaud Rahier, Houda Abla, Yani Arhab, Alexandre Noiriel, and Abdelkarim Abousalham Abstract 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 2+ 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 contin- uous way and on a microplate scale. Key words Phospholipase D, 8-Hydroxyquinoline, Assay, Fluorescence, Phosphatidic acid, Micro- plate, Transphosphatidylation, Inhibitors, Screening 1 Introduction Through its central role in membrane signaling and vesicular traf- ficking, phospholipase D (PLD) has been linked to numerous pathologies, such as cancers [1]. At a molecular level, PLD hydro- lyzes the distal phosphodiester bond of glycerophospholipids, to produce phosphatidic acid (PA) and to release the corresponding headgroup. PLD also catalyzes the transphosphatidylation reaction which, in the presence of a primary alcohol, produces the corresponding phosphatidylalcohol [2]. Although many methods for measuring PLD activities have been described to date, they all present several limitations in terms of implementation, sensitivity, or specificity of detection. So far, radiolabeled phospholipids have been the most extensively used substrates for PLD [3]. However, Georgina Sandoval (ed.), Lipases and Phospholipases: Methods and Protocols, Methods in Molecular Biology, vol. 1835, https://doi.org/10.1007/978-1-4939-8672-9_6, © Springer Science+Business Media, LLC, part of Springer Nature 2018 129