Catalytic properties of lipopolysaccharide (LPS) binding protein. Transfer of LPS to soluble CD14.
Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 (sCD14) and transfer of LPS from LPS.sCD14 complexes to HDL particles. To characterize the first of these two reactions, LPS covalently derivatized with the fluorophore, boron dipyrromethene difluoride (BODIPY), was used to monitor LBP-catalyzed movement of LPS in real time. The fluorescence efficiency of micelles of BODIPY-LPS was low but was strongly increased upon dissolution in detergent or upon binding to sCD14. Spontaneous binding of BODIPY-LPS to sCD14 was very slow but was accelerated by substoichiometric concentration of LBP, and the rate of binding was measured under a variety of conditions. LBP-catalyzed transfer was first order with respect to both sCD14 and LPS concentration, and the apparent Km values were 1 approximately 2 microg/ml for sCD14 and 100 ng/ml for LPS. The maximum turnover number for LBP was approximately 150 molecules of LPS min-1 LBP-1. LBP alone caused a small but measurable increase in the fluorescence of BODIPY-LPS, suggesting that it bound LPS aggregates but did not readily remove LPS monomers. The subsequent addition of sCD14 caused a large fluorescence increase, suggesting transfer of BODIPY-LPS to sCD14. These and other observations suggest that LPS is transferred by an ordered ternary complex reaction mechanism in which LBP transfers LPS monomer from LPS aggregates to sCD14 without dissociating from the LPS aggregate.
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Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021, USA.
Wright
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D000209:Acute-Phase Proteins; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D001896:Boron Compounds; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008823:Micelles; D012475:Salmonella; D013050:Spectrometry, Fluorescence; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D015703:Antigens, CD; D001896:Boron Compounds; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008823:Micelles; D000099231:Lipopolysaccharide-Binding Protein; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene