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

1. 从猪脑和牛脑中分别分离出一种新的膜结合的mctalloendeptidase裂解前胃泌素c -末端-(88-101)肽、缓激肽和bam12p，以及一种Ras c -末端CAAX基序特异性内肽酶及其相关蛋白，并进行了纯化和鉴定。从猪十二指肠中溶出肠肽酶，对其进行纯化，并对其碳水化合物部分进行了表征；系统地研究了其对各种合成肽底物的特异性。研究了胰蛋白酶和α -胰凝乳蛋白酶在高pH条件下的肽键特异性，以限制其裂解特异性；结果表明，在高碱性pH （pH-13）下，它们分别选择性地切割Arg-X和pH- x键。从大鼠肝脏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。大肠蛋白酶（一种来自肠的新型膜结合丝氨酸蛋白酶）在人和大鼠组织中的组织分布进行了研究。分离并测序了小鼠组织蛋白酶E基因，明确了其染色体位置。此外，我们还研究了中性pH下cethepsin E的底物特异性以及与人胃粘膜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同工型，并测定了其n端氨基酸序列。与本项目相关，我们还对其他一些蛋白酶进行了进一步的表征研究，包括戊糖苷酶、蝇虫蛋白酶、霉菌酸性蛋白酶A和B、细菌金属内肽酶和信号肽酶以及菠萝蛋白酶抑制剂。

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.