Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells.
Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.
Department of Chemistry, National Institute of Health, Tokyo, Japan.
S
S
Hara-Kuge
F
F
Amano
M
M
Nishijima
Y
Y
Akamatsu
D000818:Animals; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics*; D006090:Gram-Negative Bacteria; D007700:Kinetics; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology*; D008264:Macrophages / Q000276:immunology; D015394:Molecular Structure; D009154:Mutation; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011971:Receptors, Immunologic