| Summary: | 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.
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