Identification of Re lipopolysaccharide-binding protein on murine erythrocyte membrane.
Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.
D000818:Animals; D000937:Antigen-Antibody Reactions; D003101:Collodion; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis* / Q000378:metabolism; D014644:Genetic Variation; D005960:Glucosides; D066298:In Vitro Techniques; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011402:Pronase / Q000378:metabolism; D011971:Receptors, Immunologic / Q000032:analysis* / Q000378:metabolism; D012475:Salmonella / Q000378:metabolism*
D003902:Detergents; D005960:Glucosides; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011971:Receptors, Immunologic; C018619:octyl-beta-D-glucoside; D003101:Collodion; D011402:Pronase