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Structure-activity relationship of endothelin and characterization of its converting enzyme

内皮素的构效关系及其转化酶的表征

基本信息

批准号：
01580150
负责人：
KIMURA Sadao
金额：
$ 1.47万
依托单位：
University of Tsukuba
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

1. Structure-activity relationship study of endothelin.In order to clarify the essential structure part in the active molecule, we synthesized endothelin related peptides including endothelin-1(1-21), (1-20), (1-19), (1-16), big endothelin-1(1-39), (125), carboxymethylatedendothehn-land Lys9-nicked endothelin-1 and measured their contractile activities. From the results, we concluded that the C-terminal Trp2l and SS bridges are essential for expression of RW vasoconstrictor activity2. Isolation and determination of endothelins in the suliematant of cultured endothelial cells, spinal cord and brain.From the supernatant of cultured endothelial cells, we isolated endothelin-1(1-21), big endothelin-l(l-j9)and big endothelin-1(23-39)/(22-39). From the results, we concluded that endothelin-1 is synthesized from big endothelin-1 by endothelin converting enzyme which cleaves the bond of Trp2l-Val22. We also isolated and determined the structures of endothelin-1 and endothelin-3 from porcine spinal cord and brain. We showed that these endothelin may act as neuropeptides in central nervous system.3. Purification and characterization of endothelin converting enzyme.We purified partly and characterized the endothelin converting enzymes from bovine adrenal medulla, aortic endothelial cells and porcine lung and brain. One is an acidic protease which is found to be cathepsin-D, later. Other is a neutral metal protease. The acidic protease and the neutral protease are strongly inhibited by pepstatin and phosphoramidon, respectively. However, cathepsin D was found to be not a physiological enzyme, although it has endothelin converting activity.

1.内皮素的构效关系研究：为了阐明内皮素活性分子中的关键结构部分，我们合成了内皮素相关肽，包括内皮素-1（1-21）、（1-20）、（1-19）、（1-16）、大内皮素-1（1-39）、（125）、羧甲基化内皮素和赖氨酸9缺口内皮素-1，并测定了它们的收缩活性。从结果中，我们得出结论，C-末端Trp 2l和SS桥是必不可少的RW血管收缩活性的表达2。内皮细胞培养上清液、脊髓和脑中内皮素的分离和测定从培养的内皮细胞上清中分离出内皮素-1（1-21）、大内皮素-1（1-j 9）和大内皮素-1（23-39）/（22-39）。结果表明，内皮素-1是由大内皮素-1通过内皮素转化酶切断Trp 2l-Val 22键合成的。我们还从猪脊髓和脑中分离并测定了内皮素-1和内皮素-3的结构。提示这些内皮素可能是中枢神经系统的神经肽.内皮素转换酶的分离纯化及性质研究：我们从牛肾上腺髓质、主动脉内皮细胞、猪肺和猪脑中分离纯化了内皮素转换酶，并对其进行了部分性质研究。一种是酸性蛋白酶，后来发现是组织蛋白酶-D。另一种是中性金属蛋白酶。酸性蛋白酶和中性蛋白酶分别被胃蛋白酶抑素和磷酰胺强烈抑制。然而，发现组织蛋白酶D不是生理酶，尽管它具有内皮素转化活性。