Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification.
Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.
Wu
J S
JS
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Lever
J E
JE
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D002460:Cell Line; D006825:Hybridomas / Q000276:immunology; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008871:Microvilli / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000276:immunology / Q000378:metabolism*; D010695:Phlorhizin / Q000378:metabolism; D011485:Protein Binding; D013552:Swine
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D009004:Monosaccharide Transport Proteins; D010695:Phlorhizin