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

新型脑肠蛋白酶的结构、功能和生理学特征

基本信息

批准号：
09480153
负责人：
TAKAHASHI Kenji
金额：
$ 1.28万
依托单位：
Tokyo University of Pharmacy and Life Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

1.分别从猪脑和牛脑中提取、纯化并鉴定了一种新的膜结合型内肽酶，该酶能切割前胃泌素C-末端（88-101）肽、缓激肽和BAM-12 P，并能特异性切割Ras及其相关蛋白C-末端CAAX基序.从猪十二指肠中溶解肠肽酶，纯化并表征其碳水化合物部分;系统研究了其对各种合成肽底物的特异性.胰胰蛋白酶和α-糜蛋白酶的肽键特异性在高pH下进行了研究，以限制切割特异性;它们显示在高碱性pH（pH-13）下分别选择性切割Arg-X和Phe-X键。通过对大鼠肝微粒体膜结合丝氨酸蛋白酶的多肽链组成和部分氨基酸序列分析，确定其为hepsin.用人和大鼠的组织研究了结肠蛋白酶（一种新的来自肠道的膜结合丝氨酸蛋白酶）的组织分布.对小鼠组织蛋白酶E基因进行了分离和测序，并阐明了其染色体定位。此外，还研究了人胃粘膜中cethepsin E在中性pH下的底物特异性及其与α 2-巨球蛋白的相互作用.从腔棘鱼胃粘膜中分离并鉴定了胃蛋白酶原亚型，并测定了其N端氨基酸序列.与该项目相关，对其他一些蛋白酶进行了额外的表征研究，包括猪笼草蛋白酶、蛙蛋白酶、霉菌酸性蛋白酶A和B、细菌金属内肽酶和siganal肽酶以及菠萝蛋白酶抑制剂。