Detection of lipopolysaccharide-binding proteins on membranes of murine lymphocyte and macrophage-like cell lines.
Lipopolysaccharide-(LPS) binding proteins present on murine-lymphocyte and macrophage-like cell lines were identified by a ligand-blotting method and subsequent immunological detection of bound LPS. Membrane proteins of the murine-pre-B-cell line 70Z/3 were separated by SDS-PAGE, transferred electrophoretically onto nitrocellulose, and the blot was incubated with LPS of the Salmonella minnesota Re-mutant R595 (mRe-LPS). LPS bound to proteins on nitrocellulose was immunologically detected by anti-mRe-LPS antibodies; LPS was associated with one of the membrane proteins of 70Z/3 cells. This protein was 40 kDa under reducing and 45 kDa under non-reducing conditions, respectively. Treatment of 70Z/3 cells with pronase led to the disappearance of the LPS-binding protein indicating its surface location. Excess free lipid A, which represents the biologically active region of LPS, inhibited the binding of mRe-LPS to the protein. This LPS-binding protein was also identified on the pre-B-cell line CYG8, the B-cell line CYG101 and the murine-T-cell line BW5147. It was, however, not detectable on the B-cell line CYG34 and the myeloma-cell line P3-X63-Ag8.653. No other LPS-binding protein could be detected on these cell lines. In the murine-macrophage-like cell line J774.1, two LPS-binding proteins, one of 40 kDa and one of 80 kDa, were detected. These results indicate that mRe-LPS is specifically bound to a 40-kDa protein of lymphocytes, whereas in the case of macrophages it is associated with two LPS-binding proteins of 40 and 80 kDa.
Kirikae
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Forschungsinstitut Borstel, Institut für Experimentelle Biologie und Medizin, F.R.G.
Kirikae
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Schade
F U
FU
Yoshida
M
M
Kondo
S
S
Hisatsune
K
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Nishikawa
S
S
Rietschel
E T
ET
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000737:chemistry; D008050:Lipid A / Q000378:metabolism; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008214:Lymphocytes / Q000737:chemistry*; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000737:chemistry
D004731:Endotoxins; D008050:Lipid A; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011971:Receptors, Immunologic