Structural, Functional and Physiological Characterization of Novel Brain-Gut Proteinases

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1. A novel membrane-bound mctalloendopeptidase cleaving progastrin C-terminal-(88-101) peptide as well as bradykinin and BAM-12P and an endopeptidase specific for the C-terminal CAAX motif of Ras and related proteins were solubilized from porcine and bovine brains, respectively, purified and characterized.2. Enteropeptidase was solubilized from porcine duodenum, purified and characterized with special reference to the carbohydrate moieties ; the specificity toward various synthetic peptide substrates was sysytematically investigated.3. The peptide bond specificities of pancreatic trypsin and alpha-chymotrypsin were investigated at high pH in order to restrict the cleavage specificity ; they were shown to selectively cleave Arg-X and Phe-X bonds, respectively, at a high alkaline pH (pH-13).4. The microsomal membrane-bound serine proteinase from rat liver was identified as hepsin from analyses of its polypeptide chain composition and partial amino acid sequence.5. The tissue distribution of colopsin (a novel membrane-bound serine proteinase from intestine) was investigated with human and rat tissues.6. The gene for mouse cathepsin E was isolated and sequenced, and its chromosomal location was clarified. In addition, the substrate specificity at neutral pH and interaction with alpha 2-macroglobulin of cethepsin E from human gastric mucosa were investigated.7. Pepsinogen isoforms were isolated and characterized from the gastric mucosae of coelacanth stomach and the N-terminal amino acid sequences were determined.8. In relation to this project, additional characterization studies were performed on some other proteinases including nepenthesin, flarial proteinases, mold acid proteinases A and B, bacterial metalloendopeptidases and siganal peptidase and bromelain inhibitors.