Monoclonal Antibodies to Renal Na-Amino Acid Transporter

Mamelok, Richard

Abstract

This proposal seeks to develop a collection of monoclonal antibodies against Na+ dependent transport systems of the rabbit renal proximal tubule for neutral (L-alanine) and acidic (L-glutamate) amino acids. Brush border membranes will be prepared and used as an antigenic inoculum for BALB/c mice. Splenic cells of these immunized mice will be fused with the mouse myeloma cell line, P3/X63/AG8.653 in the presence of ethylene glycol. Antibodies which specifically inhibit Na+ dependent amino acid transport will be identified and the hybridomas producing these antibodies will be cloned. Monoclonal antibodies will be used to probe the membrane bound transport system. The number of proteins comprising a single transport system will be determined. The molecular weights and isoelectric points of the antigenic components of the transport system will be assayed respectively by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. By selectively labeling the inside or outside surface of right side out brush border vesicles with radiolabeled monoclonal antibodies, the symmetry of the transport system will be determined. The question: do the proteins involved with amino acid transport span the entire membrane? will be answered. The effect of Na+ and amino acid on the orientation and mobility of the transport protein(s) in the membrane will be probed. Selected monoclonal antibodies will be used as ligands for affinity columns to purify the components of the transport systems. These components will be inserted into the walls of liposomes in order to determine the requirements to reconstitute the purified transport system. Comparisons of the properties of the neutral and acidic amino acid transport systems will be made. Also, the binding constants of Na+ and amino acid to the purified transporter will be measured. Whether the binding sites for Na+ and amino acid are on the same or different proteins will be ascertained.