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Studies on Anti-Amnesiac Effect of Specific Inhibitor for Prolyl Endopeptidase

脯氨酰内肽酶特异性抑制剂抗遗忘作用的研究

基本信息

批准号：
01571217
负责人：
YOSHIMOTO Tadashi
金额：
$ 1.34万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Prolyl endopeptidase (EC 3.4.21.26) is widely distributed in animal tissues and seems to play some role in the metabolism of biologically active peptides. We found the potent inhibitors specific for the enzyme. Z-Gly-Pro-CH2Cl. Z-Pro-prolinal and the related compounds show an antiamnesic effect on experimental amnesia in rat and mouse induced by scopolamine or electro-convulsive shock. This antiamnesic effect seems to be produced by the inhibition of the enzyme in brain. In order to clarify the mechanism. Function relationship of the inhibitors for the enzyme was studied and the enzyme gene was cloned.1. Study on active site of prolyl endopeptidase : Structural requirements of N-blocked L-proline derivatives as specific inhibitors for prolyl endopeptidase were investigated using a series of substrate analogs. Replacement of L-proline by its D-isomer remarkably reduced the inhibition. Introduction of a sulfur atom in proline and/or in the penultimate pyrrolidine rings significantly increased the inhibition, but the introduction of Oxygen rather diminished the activity. A peptide linkage between the proline and the Pyrrolidine ring was also require to keep the inhibitory activity. A benzyloxycarbonyl group was most effective as an N-blocked component of the inhibitors.2. Cloning of prolyl endopeptidase gene : Amino acid sequence of the proteolytic peptides of the enzyme were determined by Edman degradation after isolation by HPLC. Probes for Southern analysis were prepared following the amino acid sequences. The gene encoding prolyl endopeptidase from Flavobacterium meningosepticum was isolated and the entire nucleotide sequence of the coding sequence was determined. The transformant of E. Coli DH1 harboring pFPH5K50 with a 2.5 kbp fragment showed high prolyl endopeptidase activity. By this project. The structure at active site of prolyl endopeptidase and nucleotide sequence of the enzyme gene were clarified.

脯氨酰内肽酶(EC 3.4.21.26)广泛存在于动物组织中，在生物活性多肽的代谢中起一定作用。我们发现了这种酶所特有的有效抑制剂。Z-Gly-Pro-CH2Cl.Z-Pro-Pro-prolinal及其相关化合物对大鼠和小鼠由东莨菪碱或电惊厥所致的实验性遗忘有明显的抗记忆作用。这种抗健忘作用似乎是通过抑制大脑中的酶而产生的。以期阐明其作用机制。研究了酶抑制剂的作用关系，克隆了该酶基因。Pro-内肽酶活性部位的研究：采用一系列底物类似物研究了N-封闭型L-Pro衍生物作为Pro-内肽酶特异性抑制剂的结构要求。用其D-异构体取代L-脯氨酸后，抑制作用明显减弱。在脯氨酸和/或倒数第二个吡咯环上引入硫原子显著增加了抑制作用，但氧的引入反而降低了活性。为了保持抑制活性，还需要在脯氨酸和吡咯烷环之间建立多肽键。作为抑制剂的N-封闭成分，苄氧羰基是最有效的。Pro内肽酶基因的克隆：高效液相色谱分离后，用Edman降解法测定该酶蛋白水解肽的氨基酸序列。根据氨基酸序列制备用于Southern分析的探针。从脑膜败血黄杆菌中分离出编码脯氨酰内肽酶的基因，并测定了编码序列的全序列。含有2.5kb片段pFPH5K50的E.Coli DH1转化子具有较高的脯氨酰内肽酶活性。通过这个项目。明确了该酶活性部位的结构和酶基因的核苷酸序列。