J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.D500005-JLR200 on April 1, 2005

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
D500005-JLR200v1
46/6/1339    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sabirsh, A.
Right arrow Articles by Owman, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sabirsh, A.
Right arrow Articles by Owman, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 46, 1339-1346, June 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Fluorescent leukotriene B4: potential applications

Alan Sabirsh1,*, Anders Wetterholm*, Jesper Bristulf{dagger}, Hakon Leffler§, Jesper Z. Haeggström* and Christer Owman{dagger}

* Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, Stockholm, Sweden
{dagger} Department of Physiological Sciences, Division of Molecular Neurobiology, Lund University, Lund, Sweden
§ Department of Laboratory Medicine, Division of Microbiology, Immunology, and Glycobiology, Lund University, Lund, Sweden

Published, JLR Papers in Press, April 1, 2005. DOI 10.1194/jlr.D500005-JLR200

1 To whom correspondence should be addressed. e-mail: alan.sabirsh{at}mbb.ki.se

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer.

In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Supplementary key words G-protein coupled receptor • pharmacology • method


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?




HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.