Our main objectives are to elucidate and define the biochemical and physiological mechanisms involved in the digestion and absorption of dietary protein, peptides and amino acids in the mammalian intestine. During the forthcoming grant period we plan to continue our studies with brush border membrane aminopeptidase N and dipeptidyl aminopeptidase IV that have been purified in our laboratory. These studies include a detailed structural analysis of the enzyme carbohydrate moieties and their role in enzyme stabilization and catalysis. The biosynthesis, intracellular processing and ultimate insertion into the microvillus membrane of the enzyme will be determined by use of specific monoclonal antibodies, radioimmunoassay, subcellular fractionation and various highly specific inhibitory drugs such as colchicine, actinomycin D and tunicamycin. The macromolecular arrangement and spatial relationship of these two enzymes in the microvillus membrane will be investigated using bifunctional crosslinking agents and monospecific antibody. We also plan to initiate studies that will eventually lead to the solubilization, purification and characterization of four new brush border membrane peptidases recently identified in our laboratory. These may play important roles in protein digestion. Finally, the effect of dietary components on the levels of peptidases in different mucosal cells subfractions and in various regions of the small intestine will be investigated.
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