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Journal of Lipid Research, Vol. 48, 472-482, February 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Lyndon J. Mitnaul^1,*, Jenny Tian^*, Charlotte Burton^*, My-Hanh Lam^*, Yuping Zhu, Steve H. Olson, Jonathan E. Schneeweis, Paul Zuck, Shilpa Pandit^*, Matt Anderson^*, Milana M. Maletic, Sherman T. Waddell, Samuel D. Wright^*, Carl P. Sparrow^* and Erik G. Lund^*

^* Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065
Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454

Published, JLR Papers in Press, November 7, 2006.

¹ To whom correspondence should be addressed. e-mail: lyndon_mitnaul{at}merck.com

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.

Supplementary key words bodipy-labeled • synthetic high density lipoprotein • micelles

Abbreviations: apoE, apolipoprotein E; apo HDL, purified protein component of human high density lipoprotein; BD-ffa, Bodipy-labeled free fatty acid; bis-BD-PC, bis-labeled-Bodipy-phosphatidycholine; EL, endothelial lipase; HDL-C, high density lipoprotein cholesterol; HUVEC, human umbilical vein endothelial cell; HUVEC-EL, human umbilical vein endothelial cell-derived endothelial lipase; mono-BD-TG, mono-labeled-Bodipy-triglyceride; TNF-, tumor necrosis factor-

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