Shinji Takai1, Shin-ichiro Sumi2, Masaki Aoike2,
Masato Sakaguchi1,
Yoshimi Itoh3, Denan Jin1, Eiko Matsumura3
and Mizuo Miyazaki1
1Departmentﾊof Pharmacology, Osaka Medical College, Takatsuki
City, Osaka 569-8686, Japan
2Instituteﾊfor Biotechnology Research, Wakunaga Pharmaceutical
Co., Ltd., Kodacho, Takatagun, Hiroshima 739-1195, Japan
3Departmentﾊof Cell Biology, Osaka University of Pharmaceutical
Sciences, Takatsuki City, Osaka 569-1041, Japan
Abstract: We compared recombinant human chymase expressed in
Escherichia coli with human chymase purified from vascular tissues.
The recombinant chymase, the structure of which was NH2-enterokinase
cleavage site-chymase-COOH, was expressed in Escherichia coli and
then was solubilized and renatured. The protein did not have a chymase activity,
but gained this activity after the cleavage of the N-terminal site by enterokinase.
The enzyme was purified by heparin affinity and gel filtration columns.
The N-terminal sequence of the protein was identical to the sequence for
human chymase. The molecular weights of the recombinant chymase and chymase
purified from human vascular tissues were 26 and 30ﾊkDa, respectively, and
the 4ﾊkDa difference was thought to be due to the presence or absence of
glycan. The optimum pH of the recombinant enzyme activity was between 7.5
and 9.0. The activity of the recombinant enzyme was inhibited by chymostatin,
soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by
ethylenediaminetetraacetic acid and aprotinin. This enzyme cleaved specifically
the Phe8-His9 bond of angiotensin (Ang)ﾊI to form
AngﾊII and that of big endothelin (ET)-1 to form ET-1-(1-31). These findings
demonstrated that the enzymatic characteristics of the recombinant enzyme
were identical to that of native human chymase.
Keywords: Chymase, Recombinant, AngiotensinﾊII, Inhibitor