Continuous, vesicle-based fluorimetric assays of 14- and 85-kDa phospholipases A2.

Abstract

This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A2 (cPLA2) and the 14-kDa, human nonpancreatic, secreted phospholipase A2 (sPLA2). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized and shown to be substrates for cPLA2 in a fluorescence-based assay. Most of the assays make use of small and large unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-micelles. The cPLA2 assays can be carried out in a fixed time-point mode in which on..