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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK017938-21
Application #
2137140
Study Section
Special Emphasis Panel (NSS)
Project Start
1977-09-30
Project End
1996-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
21
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Erickson, R H; Lai, R S; Lotterman, C D et al. (2000) Identification of upstream stimulatory factor as an activator of the human dipeptidyl peptidase IV gene in Caco-2 cells. Gene 258:77-84
Erickson, R H; Lai, R S; Kim, Y S (2000) Role of hepatocyte nuclear factor 1alpha and 1beta in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells. Biochem Biophys Res Commun 270:235-9
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Erickson, R H; Gum, J R; Lotterman, C D et al. (1999) Regulation of the gene for human dipeptidyl peptidase IV by hepatocyte nuclear factor 1 alpha. Biochem J 338 ( Pt 1):91-7
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Bohm, S K; Gum Jr, J R; Erickson, R H et al. (1995) Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter. Biochem J 311 ( Pt 3):835-43
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