title: A real-time fluorogenic phospholipase A(2) assay for biochemical and cellular activity measurements.
abstract: A fluorogenic analog of the PLA(2) substrate PC, named Dabcyl-BODIPY-PC, or simply DBPC, was synthesized with a fluorescence quencher (Dabcyl, 4-[(4-[N,N-dimethylamino]phenyl)azo]benzoic acid) in the sn-1 acyl chain and a BODIPY fluor in the sn-2 acyl chain. DBPC was recognized by sPLA(2) from each of the four sources examined (bee venom, human synovial fluid, cobra venom, and bovine pancreas). A dramatic and quantifiable fluorescence enhancement of DBPC occurred upon phospholipase digestion both in the presence and absence of excess PC. Both real-time and endpoint assays for PLA(2) were sensitive, consistent, and rapid. Thus, DBPC can be used as a sensitive fluorogenic probe for in vitro high-throughput screening assays for PLA(2) activation and inhibition and would expedite studies of PLA(2) in cellular signaling, in vitro screening for drug discovery, and subcellular localization of enzyme activity.
authors.affiliation: Center for Cell Signaling, 420 Wakara Way, Suite 360, Salt Lake City, UT 84108, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Feng
authors.forename: Kelly
authors.initials: K
authors.lastname: Manabe
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Shope
authors.forename: Stanton
authors.initials: S
authors.lastname: Widmer
authors.forename: Daryll B
authors.initials: DB
authors.lastname: DeWald
authors.forename: Glenn D
authors.initials: GD
authors.lastname: Prestwich
mesh_terms: D000818:Animals; D001514:Bee Venoms / Q000378:metabolism; D001896:Boron Compounds / Q000737:chemistry; D002417:Cattle; D004285:Dogs; D004546:Elapid Venoms / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000737:chemistry*; D010740:Phospholipases / Q000737:chemistry; D010741:Phospholipases A / Q000032:analysis* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000201:enzymology
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001514:Bee Venoms; D001896:Boron Compounds; D004546:Elapid Venoms; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010740:Phospholipases; D010741:Phospholipases A