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

1.内皮素构效关系的研究：为了阐明活性分子中的主要结构成分，我们合成了内皮素相关多肽，包括内皮素-1(1-21)、(1-20)、(1-19)、(1-16)、大内皮素-1(1-39)、(125)、羧甲基化内毒素-1(1-39)、(125)，并测定了其收缩活性。根据这些结果，我们得出结论，C-末端Trp21和SS桥是RW血管收缩活性表达的关键。从培养内皮细胞培养上清液、脊髓和脑组织中分离出内皮素-1(1-21)、大内皮素-L(L-J9)和大内皮素-1(23-39)/(22-39)。结果表明，内皮素-1是由大的内皮素-1通过内皮素转换酶裂解Trp21-Val22的键而合成的。我们还从猪的脊髓和脑中分离并鉴定了内皮素-1和内皮素-3的结构。提示这些内皮素可能在中枢神经系统中起神经肽的作用。我们从牛肾上腺髓质、主动脉内皮细胞和猪肺、脑中分离纯化了部分内皮素转换酶，并对其进行了鉴定。一种是酸性蛋白酶，后来被发现是组织蛋白酶-D。另一种是中性金属蛋白酶。酸性蛋白水解酶和中性蛋白水解酶分别被胃抑素和磷酰胺强烈抑制。然而，组织蛋白酶D被发现不是一种生理酶，尽管它具有内皮素转化活性。

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.