title: Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.
abstract: Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, 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 during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale.
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS) UMR 5246 CNRS, Université Claude Bernard Lyon 1, Organisation et Dynamique des Membranes Biologiques, Bâtiment Raulin, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Rahier
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS) UMR 5246 CNRS, Université Claude Bernard Lyon 1, Organisation et Dynamique des Membranes Biologiques, Bâtiment Raulin, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Noiriel
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS) UMR 5246 CNRS, Université Claude Bernard Lyon 1, Organisation et Dynamique des Membranes Biologiques, Bâtiment Raulin, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Abousalham
mesh_terms: D000438:Alcohols / Q000494:pharmacology; D010367:Arachis / Q000201:enzymology; D001937:Brassica / Q000201:enzymology; D007887:Fabaceae / Q000201:enzymology; D005453:Fluorescence; D015394:Molecular Structure; D015125:Oxyquinoline / Q000737:chemistry*; D010739:Phospholipase D / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology
chemical_list: D000438:Alcohols; D011994:Recombinant Proteins; D015125:Oxyquinoline; D010739:Phospholipase D