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

脯氨酰内肽酶（EC 3.4.21.26）广泛分布于动物组织中，似乎在生物活性肽的代谢中起一定作用。我们发现了这种酶的有效抑制剂。Z-Gly-Pro-CH2Cl。Z-脯氨酸及其有关化合物对东莨菪碱或电休克引起的大鼠和小鼠实验性遗忘有抗遗忘作用。这种抗遗忘作用似乎是通过抑制大脑中的酶而产生的。为了阐明机制。研究了该酶抑制剂的功能关系，并克隆了该酶基因.脯氨酰内肽酶活性中心的研究：利用一系列底物类似物研究了N-封闭的L-脯氨酸衍生物作为脯氨酰内肽酶特异性抑制剂的结构要求。用L-脯氨酸的D-异构体取代L-脯氨酸可显著降低这种抑制作用。在脯氨酸和/或倒数第二个吡咯烷环中引入硫原子显著增加了抑制作用，但引入氧反而降低了活性。脯氨酸和吡咯烷环之间的肽键也是保持抑制活性所必需的。苄氧羰基是最有效的N-封闭的组成部分的走廊。脯氨酰内肽酶基因的克隆：高效液相色谱法分离脯氨酰内肽酶蛋白，Edman降解法测定蛋白水解肽的氨基酸序列。按照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脯氨酰内肽酶的编码基因，并测定了其编码序列的全部核苷酸序列。对E.含有2.5 kbp片段的pFPH 5 K50的Coli DH 1显示出高的脯氨酰内肽酶活性。通过这个项目。阐明了脯氨酰内肽酶活性部位的结构和酶基因的核苷酸序列。

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.