Detection of lipopolysaccharide‐binding proteins on membranes of murine lymphocyte and macrophage‐like cell lines

Summary 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.