A novel fluorogenic substrate for the measurement of endothelial lipase activity

Endothelial lipase (EL) is a phospholipase A(1) (PLA(1)) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA(1) activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA(2) enzymes. In order to distinguish PLA(1) activity of EL from PLA(2) enzymatic activity in cell-based assays, cell supernatants, and other non-homogeneous systems, a novel fluorogenic substrate with selectivity toward PLA(1) hydrolysis was conceived and characterized. This substrate was preferred by PLA(1) enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA(2) activity. The phospholipase activity detected by the PLA(1) substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA(1) substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA(1) enzymes, such as EL, and aiding in characterizing their mechanisms of action.-Darrow, A. L., M. W. Olson, H. Xin, S. L. Burke, C. Smith, C. Schalk-Hihi, R. Williams, S. S. Bayoumy, I. C. Deckman, M. J. Todd, B. P. Damiano, and M. A. Connelly. A novel fluorogenic substrate for the measurement of endothelial lipase activity. J. Lipid Res. 2011. 52: 374-382