Fluorogenic phospholipids as head group-selective reporters of phospholipase A activity.

PLA (phospholipases A) are important mediators of cell signaling, generating bioactive fatty acids and LPLs (lysophospholipids). PLA products having different head groups can initiate vastly different types of signaling. Fluorogenic analogues of the PLs (phospholipids) PA (phosphatidic acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), and PG (phosphatidylglycerol) were synthesized as PLA substrates for rapidly determining in real time the influence of head group modifications on cell signaling both in vitro and in cells. Enzyme-assisted remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom PLA 2 and transphosphatidylation with a particular PLD (phospholipase D) were central steps in the preparation of these enzymatic probes. The resulting fluorogenic Dabcyl- and BODIPY-containing PL analogues, DBPA, DBPC, DBPE, and DBPG, were used in mixed micelle assays to determine PLA 2 kinetics. Next, the assays were used to determine the X i (50) value of a common PLA 2 inhibitor. Finally, the head group selectivities of a series of commercially available PLA 2 enzymes were readily established using the DBPLs (Dabcyl-BODIPY PLs) as substrates.