title: A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities.
abstract: A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.
authors.affiliation: Univ Lyon, Université Lyon 1, UMR 5246, CNRS, INSA Lyon, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Métabolismes, Enzymes et Mécanismes Moléculaires (MEM), F-69622 Villeurbanne, France Univ Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Chimie Organique et Bioorganique (COB), F-69621 Villeurbanne, France.
authors.forename: Meddy
authors.initials: M
authors.lastname: El Alaoui
authors.affiliation: Univ Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Chimie Organique et Bioorganique (COB), F-69621 Villeurbanne, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Soulère
authors.affiliation: Univ Lyon, Université Lyon 1, UMR 5246, CNRS, INSA Lyon, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Métabolismes, Enzymes et Mécanismes Moléculaires (MEM), F-69622 Villeurbanne, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Noiriel
authors.affiliation: Univ Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Chimie Organique et Bioorganique (COB), F-69621 Villeurbanne, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Popowycz
authors.affiliation: Univ Lyon, Université Lyon 1, UMR 5246, CNRS, INSA Lyon, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Métabolismes, Enzymes et Mécanismes Moléculaires (MEM), F-69622 Villeurbanne, France.
authors.forename: Abdallah
authors.initials: A
authors.lastname: Khatib
authors.affiliation: Univ Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Chimie Organique et Bioorganique (COB), F-69621 Villeurbanne, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Queneau
authors.affiliation: Univ Lyon, Université Lyon 1, UMR 5246, CNRS, INSA Lyon, CPE Lyon, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), Métabolismes, Enzymes et Mécanismes Moléculaires (MEM), F-69622 Villeurbanne, France abousalham@univ-lyon1.fr.
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Abousalham
mesh_terms: D000818:Animals; D001514:Bee Venoms / Q000201:enzymology; D001516:Bees / Q000201:enzymology; D057075:Enzyme Assays; D019476:Insect Proteins / Q000737:chemistry; D008042:Linolenic Acids / Q000737:chemistry; D010713:Phosphatidylcholines / Q000096:biosynthesis / Q000737:chemistry; D054466:Phospholipases A1 / Q000737:chemistry*; D054467:Phospholipases A2 / Q000737:chemistry*; D034421:Sus scrofa
chemical_list: D001514:Bee Venoms; D019476:Insect Proteins; D008042:Linolenic Acids; D010713:Phosphatidylcholines; C004934:eleostearic acid; D054466:Phospholipases A1; D054467:Phospholipases A2
keywords: 1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine; 1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine; tung oil; α-eleostearic acid; β-cyclodextrin