Immunological approaches to the study of membrane receptors. A monoclonal antibody that inhibits the binding of asialoglycoproteins to the rat liver receptor.
The major polypeptide (43,000 daltons) of the rat liver receptor for asialoglycoproteins was isolated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Antibodies raised in a goat against this SDS-treated polypeptide exhibited marked cross-reactivity toward the SDS-denatured forms of the two other prominent polypeptides (54,000 and 64,000 daltons) of the receptor preparation. Monoclonal antibodies directed against the receptor were prepared using the spleen cells of mice immunized with the soluble, active receptor purified by affinity chromatography. The most extensively characterized of the monoclonal antibodies, designated D3-5D3, recognized the solubilized receptor and bound to the exterior surface of isolated rat hepatocytes. The binding of D3-5D3 to hepatocytes prevented subsequent binding of the ligand, 125I-asialo-orosomucoid. Conversely, occupation of the receptor with ligand inhibited binding of 125I-IgG prepared from D3-5D3 ascites fluid. The secondary structure of the receptor appears to be critical for recognition by D3-5D3, since denaturation of the receptor with 1% SDS, 5% beta-mercaptoethanol at 100 degrees C abolished antibody binding. Under less denaturing conditions (0.1% SDS, 25 degrees C), antigenic reactivity was retained by the receptor. Preparative electrophoresis using the latter conditions permitted the demonstration that D3-5D3 recognized a unique determinant that is present in each of the three polypeptides.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D002462:Cell Membrane / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D007074:Immunoglobulin G; D008099:Liver / Q000378:metabolism*; D009961:Orosomucoid / Q000031:analogs & derivatives; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D006023:Glycoproteins; D007074:Immunoglobulin G; D009961:Orosomucoid; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic