Purification and characterization of a kinin- and angiotensin II-forming enzyme in the dog heart.
OBJECTIVE
To purify and characterize a kinin-forming enzyme in the dog heart and to examine the ability of this enzyme to generate angiotensin (Ang) II from Ang I.


METHODS
The enzyme was isolated from heart homogenate using a diethylaminoethyl-Sepharose column, an aprotinin affinity column and a wheat germ lectin-Sepharose 6MB column. Kininogenase activity was assessed with a kinin radioimmunoassay after samples had been incubated with bovine low-molecular-mass kininogen at 37 degrees C for 1 h. Ang I-converting activity was assessed by the quantitation of Ang II formed by incubation of the sample with Ang I at 37 degrees C for 3 h, using high performance liquid chromatography. The enzyme was subjected to 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue and transferred electrically to a membrane with glycoprotein staining.


RESULTS
The purified enzyme is a glycoprotein with an apparent relative molecular mass of 65 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its kininogenase activity was approximately 20 micrograms bradykinin/h per mg protein at an optimal pH of 8.0. The enzyme also converted Ang I to Ang II at an optimal pH of 6.5. Its specific activity was approximately 2 micrograms Ang II/h per mg protein. Both activities were inhibited by aprotinin, a tissue kallikrein inhibitor. Western blot analysis using polyclonal antibody against this enzyme demonstrated that this enzyme exists both in the myocardium and in the coronary artery.


CONCLUSIONS
The present study showed that the kinin-forming enzyme in the dog heart is a kallikrein-like enzyme that is different from cathepsin D, cathepsin G and chymase. It is also able to Ang I to Ang II. This enzyme might play a role in regulating myocardial perfusion, mainly by generating kinins and in part by forming Ang II.
Sasaguri
M
M
Department of Internal Medicine, Fukuoka University, School of Medicine, Japan.
Maeda
H
H
Noda
K
K
Tsuji
E
E
Kinoshita
A
A
Ideishi
M
M
Ogata
S
S
Arakawa
K
K
D000804:Angiotensin II / Q000096:biosynthesis*; D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D002417:Cattle; D002847:Chromatography, Agarose; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007610:Kallikreins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007705:Kinins / Q000096:biosynthesis*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D006023:Glycoproteins; D007705:Kinins; D000804:Angiotensin II; D007611:Aprotinin; D007703:Peptidyl-Dipeptidase A; D007610:Kallikreins