Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands

Neutrophils are activated by both paracrine molecules, e.g. platelet activa ting factor (PAF) and leukotriene B-4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in re gulation of the activation, including receptor endocytosis and ligand break down. The interactions between the specific granule protein neutrophil gela tinase-associated lipocalin (NGAL), expressed in human neutrophils, and fML P, PAF and LTB4, were investigated by weak affinity chromatography. NGAL wa s immobilised to a silica matrix and packed in a micro-column and the reten tion times of retarded ligands were measured and used to calculate the stre ngth of the interactions. The association constants for fMLP were K-ass = 0 .85 . 10(3) M-1 at 20 degrees C and 0.77 . 10(3) M-1 at 37 degrees C, for L TB4 were K-ass = 4.37 . 10(3) M-1 at 20 degrees C and 3.27 . 10(3) M-1 at 3 7 degrees C and for PAF were K-ass = 25.4 . 10(3) M-1 at 20 degrees C and 1 0.5 . 10(3) M-1 at 37 degrees C. Other methods of detecting the interaction s such as gel filtration, immunoprecipitation, photoactivated ligands and f luorescence quenching proved to be insufficient. The results demonstrate th e superiority of weak affinity chromatography as a method of studying the i nteractions of the specific granule protein NGAL. (C) 1999 Elsevier Science B.V. All rights reserved.