title: High-throughput assay of secreted phospholipases A₂ inhibitors.
abstract: Attempts to characterize, quantify, and/or modulate the activity of the secreted phospholipase A(2) family of enzymes result from the diversity of physiological roles for which these enzymes have been implicated. The 1-palmitoyl-2-(10-pyrenedecanoyl)-phosphatidylglycerol (pyrenePG)-based fluorometric assay is a sensitive and readily adaptable method for further elucidating phospholipase function under various experimental conditions, as well as a tool for screening chemical libraries for potent inhibitors of this enzymatic activity. This assay is based on the observed difference in fluorescent emission of pyrene aggregated in vesicles compared to sequestered in monomeric form by binding to bovine serum albumin after lipolytic activity, thus allowing direct quantification of hydrolyzed fatty acids by the measurement of the corresponding monomeric emission intensity. The assay can be carried out in multiwell plates for high-throughput screening of compound libraries.
authors.affiliation: Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Wallace
authors.initials: W
authors.lastname: Thompson
authors.forename: Rob C
authors.initials: RC
authors.lastname: Oslund
authors.forename: James
authors.initials: J
authors.lastname: Bollinger
authors.forename: Heather
authors.initials: H
authors.lastname: Ewing
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D057166:High-Throughput Screening Assays; D006868:Hydrolysis; D007700:Kinetics; D054497:Phospholipases A2, Secretory / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011721:Pyrenes; D054852:Small Molecule Libraries; D012710:Serum Albumin, Bovine; D054497:Phospholipases A2, Secretory