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

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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.