Purification of lipopolysaccharide-binding protein from bovine serum.
Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.
Bochsler
P N
PN
Department of Pathology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901, USA.
Yang
Z
Z
Murphy
C L
CL
Carroll
R C
RC
D000209:Acute-Phase Proteins / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001780:Blood Coagulation Tests / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D013925:Thromboplastin / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D013925:Thromboplastin; D000099231:Lipopolysaccharide-Binding Protein