A real-time fluorogenic phospholipase A(2) assay for biochemical and cellular activity measurements.
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.
Feng
Li
L
Center for Cell Signaling, 420 Wakara Way, Suite 360, Salt Lake City, UT 84108, USA.
Manabe
Kelly
K
Shope
Joseph C
JC
Widmer
Stanton
S
DeWald
Daryll B
DB
Prestwich
Glenn D
GD
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
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