Hydrolysis of substance P and its analogs by angiotensin-converting enzyme from rat lung. Characterization of endopeptidase activity of the enzyme.
Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.
Yokosawa
H
H
Endo
S
S
Ohgaki
Y
Y
Maeyama
J
J
Ishii
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008297:Male; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D051381:Rats; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism*
D013373:Substance P; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A