The main objectives of this proposal are to elucidate and define the molecular, biochemical and physiological mechanisms involved in the digestion and absorption of dietary protein, peptides and amino acids in the mammalian small intestine. These studies will primarily focus on the digestive peptidases associated with the brush border membrane of small intestinal enterocytes. Several newly discovered neutral endopeptidases and proline hydrolyzing enzymes will be purified and characterized from rat intestine. Their mode of biosynthesis, intracellular processing and insertion into the microvillus membrane will be examined using specific antibodies, subcellular fractionation techniques and various inhibitory drugs. In addition the detailed structural characterization of the carbohydrate moieties of these glycoenzymes will be carried out using lectin affinity columns, glycosidase digestion, gel filtration and HPLC techniques. The possible role of the oligosaccharide moiety in enzyme stabilization, catalysis and """"""""targeting"""""""" to the brush border membrane will be evaluated. The regulatory effect of various types of diets on peptidase levels will be examined. In vivo intestinal perfusion of peptides and proteins through segments of rat intestine will be used to determine the physiological significance of the enzymes. Studies will be initiated to purify and characterize aminopeptidase N and dipeptidyl aminopeptidase IV from human intestine. Using a human colon cancer cell line (CaCo-2) as a model, the biosynthesis of these two enzymes will be examined during cellular differentiation. Finally, cDNA's for several enzymes will be identified, isolated and sequenced and used as probes to study the related mRNAs expressed in intestinal cells during dietary regulation and cellular differentiation.
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