The overall objective of this proposal is to gain a better understanding of the mechanisms of peptide transport in small intestine and kidney because peptides are nutritionally as important as amino acids. Experiments outlined in this proposal will permit us for the first time to study peptide transport in purified brush border as well as basal-lateral membrane vesicles free of cellular metabolism. Our experiments are designed to study the differences between amino acid and peptide transport systems with regard to ion requirements, solubilization using papain and detergents, and mutual interaction during transport. Our preliminary data show that the Na+ gradient hypothesis of amino acid and sugar transport across the brush border membrane is not applicable to peptide transport. The presence of single or multiple peptide transport mechanisms will be investigated by inhibition studies using a large variety of di- and tripeptides with native and reconstituted peptide transport systems. A current hypothesis is that the brush border peptidase(s) might function as carrier(s) in the translocation, as well as in the subsequent hydrolysis of peptides. The brush border membrane protein(s) essential for the transport of peptides will be selectively solubilized with detergents and the transport system reconstituted into liposomes. The reconstituted liposomes will be used as an assay system to purify the peptide carrier protein(s) to homogeneity. Studies on the nature of this protein(s) would help us to resolve the role of brush border peptidase(s) in peptide transport. Experiments on the effect of membrane phosphorylation and dephosphorylation on the transport of sugars, amino acids and peptides would give very useful information on the regulation of these transport systems by cellular processes. Since these experiments will be carried out using brush border as well as basal-lateral membranes, we will have a better understanding of the overall process of peptide absorption -- entry of peptides into the cell across the brush border membrane from the lumen, and exit from the cell of the peptides or its hydrolytic products into portal blood across the basal-lateral membrane.
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