Lipid A binding sites in membranes of macrophage tumor cells

• Published in: The Journal of biological chemistry 1988-10, Vol.263 (29), p.14802-14807
• Main Authors: Hampton, R Y, Golenbock, D T, Raetz, C R
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Cell Line; Cell Membrane - metabolism; Cell Nucleus - metabolism; Endotoxins - isolation & purification; Endotoxins - metabolism; Fundamental and applied biological sciences. Psychology; Kinetics; lipid A; Lipid A - isolation & purification; Lipid A - metabolism; macrophages; Macrophages - metabolism; Microbiology; Molecular Weight; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Phosphorus Radioisotopes
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)68109-8

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4′-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4′-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4′-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.