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