J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.D600041-JLR200 on November 7, 2006

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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. Mitnaul1,*, Jenny Tian*, Charlotte Burton*, My-Hanh Lam*, Yuping Zhu{dagger}, Steve H. Olson{dagger}, Jonathan E. Schneeweis§, Paul Zuck§, Shilpa Pandit*, Matt Anderson*, Milana M. Maletic{dagger}, Sherman T. Waddell{dagger}, Samuel D. Wright*, Carl P. Sparrow* and Erik G. Lund*

* Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065
{dagger} 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-{alpha}, tumor necrosis factor-{alpha}


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