Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands.
Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.
Bratt
T
T
The Granulocyte Research Laboratory, Department of Hematology, Finsen Centre, Rigshospitalet, Copenhagen, Denmark. tob@ssi.dk
Ohlson
S
S
Borregaard
N
N
D000209:Acute-Phase Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D007975:Leukotriene B4 / Q000494:pharmacology; D008024:Ligands; D000071068:Lipocalin-2; D054834:Lipocalins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000378:metabolism*; D015513:Oncogene Proteins; D010972:Platelet Activating Factor / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D013050:Spectrometry, Fluorescence
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C489506:LCN2 protein, human; D008024:Ligands; D000071068:Lipocalin-2; D054834:Lipocalins; D015513:Oncogene Proteins; D010972:Platelet Activating Factor; D011518:Proto-Oncogene Proteins; D007975:Leukotriene B4; D009240:N-Formylmethionine Leucyl-Phenylalanine; D018093:Gelatinases