title: Development of a high-throughput assay for measuring phospholipase A activity using synthetic 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine coated on microtiter plates.
abstract: To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen phospholipase A (PLA) activities. With the aim of developing a convenient, specific, sensitive, and continuous new ultraviolet (UV) spectrophotometric assay for PLA, we have synthesized a specific glycerophosphatidylcholine (PC) esterified at the sn-1 and sn-2 positions, with α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the glycerophospholipids harboring it. This coated PC film cannot be desorbed by the various buffers used during PLA assays. Following the action of PLA at the oil-water interface, α-eleostearic acid is freed and desorbed from the film and then solubilized with β-cyclodextrin. The UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water-soluble state. The PLA activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of absorption at 272 nm, which was found to be linear with time and proportional to the amount of added PLA. This continuous high-throughput PLA assay could be used to screen new PLA and/or PLA inhibitors present in various biological samples.
authors.affiliation: Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS) UMR 5246 CNRS, Organisation et Dynamique des Membranes Biologiques, Université Lyon 1 , Bâtiment Raulin, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
authors.forename: Meddy
authors.initials: M
authors.lastname: El Alaoui
authors.forename: Alexandre
authors.initials: A
authors.lastname: Noiriel
authors.forename: Laurent
authors.initials: L
authors.lastname: Soulère
authors.forename: Lucie
authors.initials: L
authors.lastname: Grand
authors.forename: Yves
authors.initials: Y
authors.lastname: Queneau
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Abousalham
mesh_terms: D031284:Aleurites / Q000737:chemistry; D000818:Animals; D001203:Ascomycota / Q000201:enzymology*; D001516:Bees / Q000201:enzymology*; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D008042:Linolenic Acids / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D010938:Plant Oils / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008042:Linolenic Acids; D010713:Phosphatidylcholines; D010938:Plant Oils; D047392:beta-Cyclodextrins; C004934:eleostearic acid; D010741:Phospholipases A; C031215:betadex