The long-term objectives are to elucidate mechanisms of small intestinal transport of peptides, using purified brush border membrane vesicles from mouse and human small intestine. The brush border membrane vesicles are devoid of cytoplasmic peptidase activity and can be treated with papain to remove oligopeptidases located on the membrane. Studies with brush border membrane vesicles are designed to delineate transport characteristics for peptides in the absence of peptide hydrolysis, and will be carried out with radioactive peptides. Dipeptides containing representative acidic, basic and neutral amino acids will be used as substrates to determine the transport characteristics of peptides in relation to the charge on the amino acids. The transport of tri- and tetrapeptides will be investigated in order to determine the maximum size of peptide transported across the membrane. The mechanism of energization of peptide transport will be investigated by studying the effects of membrane potential, proton gradient, and intravesicular K+ individually and in combination. The electrogenic nature of the peptide transport system will be evaluated by monitoring the fluorescence quenching of a potential-sensitive dye. The symport of protons with peptides will be directly investigated by determining the alterations in pH using acridine orange, weak organic bases and acids. Kinetic characteristics of transport such as carrier-affinity and maximal velocity will be determined and compared with those for free amino acids. Kinetic studies on mutual inhibition between peptides will be carried out to test for multiplicity of peptide transport systems. Selected di- and tripeptides containing D-amino acids will be used to determine the stereospecificity of peptide transport system. The effects of various peptides on influx of Na+ will be measured in order to test the hypothesis that there is a non-mutual stimulation of transport of Na+ by peptides. Using human small intestinal brush border membrane vesicles, transport pathways for free amino acids will be delineated by studying transport characteristics of individual amino acids and inhibitory interactions between amino acids. These studies on mechanisms of intestinal transport of peptides and amino acids using human small intestine are of great importance in our understanding of protein nutrition in health and disease.
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