Kininase activity in human platelets: cleavage of the Arg1-Pro2 bond of bradykinin by aminopeptidase P.
A proline-specific peptidase aminopeptidase P (APP, EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin was isolated from human platelets by liquid chromatography. The enzyme was purified 557 times. The native molecule has a M(r) of 223,000. Human platelet APP exists as a trimer with a subunit M(r) of 71,000. The apparent Km of platelet APP is 66 mumol/L for bradykinin and 47 mumol/L for the internally quenched fluorogenic substrate Lys (2,4-dinitrophenyl)-Pro-Pro-NH-CH2-CH2-NH-2-aminobenzoyl. 2HCl which is used for the routine determination of the enzyme activity. The optimum pH for hydrolysis of the fluorogenic substrate is 8.0, and the optimum temperature is 43 degrees. Platelet APP is inhibited by 1,10-phenanthroline and activated by Mn2+, thus confirming its metalloprotease nature. Cu2+, Zn2+ and Hg2+ are strongly inhibitory. Inhibition by cysteine protease inhibitors suggests the presence of a thiol group essential for enzymatic activity. Serine protease inhibitors do not affect the enzyme activity.
Vanhoof
G
G
Faculty of Medicine, University of Antwerp, Wilrijk, Belgium.
De Meester
I
I
Goossens
F
F
Hendriks
D
D
Scharpé
S
S
Yaron
A
A
D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001120:Arginine; D001792:Blood Platelets / Q000201:enzymology*; D001920:Bradykinin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D005029:Ethylenediamines / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D011392:Proline; D013379:Substrate Specificity; D013696:Temperature
C053143:(2-aminobenzoyl-epsilon-2,4-dinitrophenyllysyl-prolyl-prolyl)ethylenediamine; D005029:Ethylenediamines; D009842:Oligopeptides; D001120:Arginine; D011392:Proline; D010450:Endopeptidases; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; C032615:bradykinin kininase; D001920:Bradykinin