title: 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.
authors.affiliation: Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065, USA. lyndon_mitnaul@merck.com
authors.forename: Lyndon J
authors.initials: LJ
authors.lastname: Mitnaul
authors.forename: Jenny
authors.initials: J
authors.lastname: Tian
authors.forename: Charlotte
authors.initials: C
authors.lastname: Burton
authors.forename: My-Hanh
authors.initials: MH
authors.lastname: Lam
authors.forename: Yuping
authors.initials: Y
authors.lastname: Zhu
authors.forename: Steve H
authors.initials: SH
authors.lastname: Olson
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Schneeweis
authors.forename: Paul
authors.initials: P
authors.lastname: Zuck
authors.forename: Shilpa
authors.initials: S
authors.lastname: Pandit
authors.forename: Matt
authors.initials: M
authors.lastname: Anderson
authors.forename: Milana M
authors.initials: MM
authors.lastname: Maletic
authors.forename: Sherman T
authors.initials: ST
authors.lastname: Waddell
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Wright
authors.forename: Carl P
authors.initials: CP
authors.lastname: Sparrow
authors.forename: Erik G
authors.initials: EG
authors.lastname: Lund
mesh_terms: D000818:Animals; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D008049:Lipase / Q000097:blood* / Q000378:metabolism*; D051379:Mice; D015394:Molecular Structure
chemical_list: D005456:Fluorescent Dyes; C410382:LIPG protein, human; D008049:Lipase