The purpose of this proposal is to understand on a molecular level the process of organic solute/Na cotransport. Brush border membrane vesicles from the rabbit small intestine will be used as the source of the Na/glucose and Na/proline carriers. A protein conformational probe, FITC whose fluorescence is probe-environment sensitive, and a procedure for selective binding of this probe to the Na/glucose and Na/proline carriers have been developed. The probe undergoes a Na-dependent fluorescense quenching indicating a change in carrier conformation with Na binding to the carriers. The probe fluorescence response is sensitive to reagents which block the carriers' Na sites suggesting that the fluorescence response is due to Na interaction with the carrier. Binding of FITC to the carriers is irreversible, thus allowing the identification of the glucose/proline binding sites and carriers on polyacrylamide gels. A single 75,000 dalton peptide responds to Na/glucose protection indicating that this polypeptide is the glucose carrier. Similiarily, a single polypeptide responds to Na and proline consistent with a 95,000 dalton peptide being the Na/proline carrier. The physical structure of the carriers will be explored using fluorescence energy transfer between the carrier sites, from a sulfydryl group and a tryptophan to these sites and using fluorescence quenching and fluorescence polarization. The carriers will also be isolated and similiar studies performed to determine the subunit structure of these carriers. With a rapid flow apparatus the time course of the Na-induced conformational changes will be determined. These experiments will aid to our understanding of carriers and the mechanisms involved in changes in glucose/proline affinity by Na. Together with the kinetic data describing Na/proline and Na/glucose translocation, this proposal will more completely describe carrier mediated cotransporters.
