and/or aims): This is the second revision of a renewal application to study protein nutrition, in particular peptide transport in gut and kidney. The present application deals with the basic science aspects of the transport system for small peptides in the intestine, with the major emphasis on the definition of the molecular properties of the proteins responsible for transport activity i.e. the transporters. The proposal also addresses the regulation of the peptide transport function under physiological conditions. The three specific aims are to define (1). the chemical nature of the peptide transporter (2).the role of the Na/H exchanger in transporter function and (3). the regulation of peptide transport activity by hormones (thyroxine, glucocorticoids, insulin and epidermal growth factor (EGF). The studies will be conducted with isolated cells, purified membranes and purified proteins. Transport activity will be measured by following the influx of radioactivity from labelled peptides (glycyl-sarcosine) which are relatively resistant to hydrolysis by cellular peptidases. Since it is likely that multiple peptide transport systems exist in intestinal and renal brush border membranes, different radiolabelled or fluorescent probes will be used to identify the polypeptides constituting the transport systems. To purify transporter components, photoaffinity substrate binding and/or reconstitution into liposomes will be used as the assay system. Studies on hormonal regulation will be conducted in isolated cells. The activities of the peptide transporter and the Na/H exchanger will be assayed in control and hormone treated cells and also in brush border membrane vesicles isolated from these cells. Since peptide transport is unique, because of its dependence on a proton gradient rather than a sodium gradient as the energy source, the applicant believes that characterization of this transport system at the molecular level will make a substantial contribution to the field of transport of organic solutes across biological membranes.
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