PT - JOURNAL ARTICLE AU - S Charleson AU - P Prasit AU - S Léger AU - J W Gillard AU - P J Vickers AU - J A Mancini AU - P Charleson AU - J Guay AU - A W Ford-Hutchinson AU - J F Evans TI - Characterization of a 5-lipoxygenase-activating protein binding assay: correlation of affinity for 5-lipoxygenase-activating protein with leukotriene synthesis inhibition. DP - 1992 May 01 TA - Molecular Pharmacology PG - 873--879 VI - 41 IP - 5 4099 - http://molpharm.aspetjournals.org/content/41/5/873.short 4100 - http://molpharm.aspetjournals.org/content/41/5/873.full SO - Mol Pharmacol1992 May 01; 41 AB - A binding assay has been developed to measure the affinity of leukotriene synthesis inhibitors for 5-lipoxygenase-activating protein (FLAP), using human leukocyte membranes as the source of FLAP and a radioiodinated leukotriene synthesis inhibitor, 125I-L-691,831, as ligand. Linearity of specific binding of radiolabeled ligand was demonstrated with increasing protein and ligand concentrations. Saturation analysis of radioligand binding showed a Kd of 6 nM and a Bmax that, depending on the membrane preparation, varied between 8 and 53 pmol/mg of protein. An excellent correlation was shown between affinity for FLAP in the binding assay and inhibition of leukotriene synthesis in human polymorphonuclear leukocytes for compounds from two structurally distinct classes, namely indoles and quinolines. A large number of membrane-active compounds did not compete with 125I-L-691,831 binding to FLAP. In addition, direct 5-lipoxygenase inhibitors and a selection of eicosanoids were unable to compete for FLAP binding. This study validates a selective binding assay for leukotriene synthesis inhibitors whose protein target is FLAP.