The assay, purification and characterization of angiotensin con- verting enzyme.

• Main Authors: Chen, Hui-Ling, 陳慧玲
• Other Authors: Wang, Hsi-Hua; Tsai, Hsin
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/72464575638581221016

博士 === 國立臺灣大學 === 農業化學系 === 84 === A group of fluorogenic tripeptide substrates for angiotensin con- verting enzyme (ACE) has been synthesized. Tryptophan and EDANS, which act as fluorophore and quencher, respectively, were incor- porated into the synthetic substrates, so that after the C-termi- nal dipeptide being cleaved by ACE, an increase in fluorescence intensity at the tryptophan emission wavelength of 360nm was ob- served as a result of intramolecular resonance energy transfer. The tripeptides were synthesized by automatic peptide synthesizer and then followed by manual synthesis in which the succinyl and EDANS group were attached to the tripeptide-resin by amide link- ages. Among six substrates, EDANS-Suc-Phe-Trp-Leu had the lowest Km value and EDANS-Suc-Phe-His-Trp had the highest kcat value. ACE was purified from pig lung in this study. The purification was carried out by a combination of ammonium sulfate precipita- tion, ion exchange chromatography on Fractogel DEAE, affinity chromatography on ConA Sepharose, affinity chromatography on EAH -Sepharose-captopril and gel filtration on Sephacryl S-300. This purification procedure gave 46% yield of enzyme activity and 20mg of homogeneous ACE which could be used for crystallization exper- iments. The deglycosylated ACE appeared to be hydrophobic and in- active after being treated with glycosidase F at 37℃ for 6 hours , and the resulting ACE protein could not be crystallized. The native ACE, however, yielded several forms of crystals. Electron microscopic study showed that ACE molecule was featured by a dis- tinct biparitculate organization with certain degree of rotation- al and bending flexibility. This seems to be a major reason why it is so difficult to obtain high quality crystal from ACE. The deglycosylated ACE could be specifically cleaved into two halves by trypsin. It is hoped that this N domain protein will be more suitable for crystallization experiments.