A real-time fluorogenic phospholipase A2 assay for biochemical and cellular activity measurements

被引：39

作者：

Feng, L

Manabe, K

Shope, JC

Widmer, S

DeWald, DB

Prestwich, GD

机构：

[1] Univ Utah, Dept Med Chem, Salt Lake City, UT 84108 USA

[2] Ctr Cell Signaling, Salt Lake City, UT 84108 USA

[3] Utah State Univ, Dept Biol, Logan, UT 84322 USA

来源：

CHEMISTRY & BIOLOGY | 2002年 / 9卷 / 07期

关键词：

D O I：

10.1016/S1074-5521(02)00168-0

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：795 / 803

页数：9

共 37 条

[1] Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway -: Proteolytic fragment of type IVA cytosolic phospholipase A2α inhibits stimulus-induced arachidonate release, whereas that of type VICa2+-independent phospholipase A2 augments spontaneous fatty acid release [J].