Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Abstract

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.-Mitnaul, L.

Plasma Activity of Endothelial Lipase Impacts High-Density Lipoprotein Metabolism and Coronary Risk Factors in Humans

“…The reaction was started by adding 10 μl/well of a fluorogenic crude phospholipase A substrate, Bis-BODIPY FL C11-PC (B7701, Invitrogen, Carlsbad, CA) to a final working concentration of 0.02 mmol/L 23) . After 10 minutes of pre-incubation at 37 ℃, the fluorescence intensity (FLI) was continuously monitored using the Fluoroskan Ascent FL (Thermo LabSystems, Cambridge, UK) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm for 21 minutes at 37 ℃.…”

A novel fluorogenic substrate for the measurement of endothelial lipase activity

“…Other similar fl uorescent substrates have been described and used with phospholipid or TG micelles and with synthetic HDL particles in cell-free assays ( 38 ). We saw no particular requirement to incorporate the PLA 1 substrate characterized here into micelles as it could be used directly in either cell-free or cell-based assays.…”

High‐Throughput Screening of Marine Resources

“…Fluorescent, such as fluorescence resonance energy transfer, (FRET; Mere et al, 1999;Zhu, Fu, and Luo, 2012), dissociation-enhanced lanthanide fluorescent immunoassay (DEL-FIA; Newbatt et al, 2013), fluorescence polarization (Owicki, 2000), time resolved-FRET (TR-FRET), homogeneous time-resolved fluorescence (HTRF; Degorce et al, 2009;von Ahsen et al, 2006), fluorogenic substrates (Mitnaul et al, 2007), calcium flux (Luo et al, 2011), and membrane translocation (Vijayakumar, Ajay, and Bhat, 2010). Luminescent, such as bioluminescence resonance energy transfer (BRET; Boute, Jockers, and Issad, 2002), bioluminescent substrates (Yasgar et al, 2010), or luciferase reporter gene (Siebring-van Olst et al, 2013).…”