Lipid A binding sites in membranes of macrophage tumor cells.
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.
Hampton
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RY
Department of Biochemistry, University of Wisconsin-Madison 53706.
Golenbock
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Raetz
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CR
D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004731:Endotoxins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008050:Lipid A / Q000302:isolation & purification / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes
D001426:Bacterial Proteins; D004731:Endotoxins; D008050:Lipid A; D010761:Phosphorus Radioisotopes; C027315:lipid A-associated protein, Bacteria