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

1.分别从猪和牛脑中分离、纯化和鉴定了一种新的膜结合的裂解孕激素C端(88-101)肽、缓激肽和BAM-12p的膜结合的巨噬细胞内肽酶和Ras C端CAAX基序的内肽酶及其相关蛋白。从猪十二指肠中分离、纯化和鉴定了肠肽酶，并系统研究了其对不同合成肽底物的专一性。为了限制切割特异性，研究了高pH条件下胰腺胰蛋白酶和α-糜蛋白酶的肽键特异性；在高碱性pH(pH-13)条件下，它们分别选择性地切割Arg-X和Phe-X键。通过对大鼠肝脏微体膜结合丝氨酸蛋白酶的多肽链组成和部分氨基酸序列的分析，确定其为肝素。研究了一种新的肠道膜结合型丝氨酸蛋白酶Colopsin在人和大鼠组织中的分布。对小鼠组织蛋白酶E基因进行了分离和测序，明确了其染色体定位。此外，还研究了人胃粘膜组织蛋白酶E在中性pH条件下的底物专一性及其与α2-巨球蛋白的相互作用。从腔棘鱼胃粘膜中分离和鉴定了胃蛋白酶原亚型，并测定了其N-端氨基酸序列。与该项目有关，还对其他一些蛋白酶进行了特性研究，包括Nepenthein、花蕾蛋白水解酶、霉菌酸性蛋白酶A和B、细菌金属内肽酶以及Siganal多肽酶和菠萝蛋白酶抑制剂。

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.