A micromethod for the determination of the activities of kininases in rat plasma. Kinetics and inhibitory characteristics.
Kininase II (EC 3.4.15.1) (KII) and kininase I (KI) (EC 3.4.12.7) activities of rat plasma were characterized by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL), hippuryl-L-arginine (HLA) [expressed as carboxypeptidase N1 (CN1) activity] and hippuryl-L-lysine (HLL) [expressed as carboxypeptidase N2 (CN2) activity]. Using a spectrophotometric assay, biochemical characteristics of the three enzymes were investigated. The Michaelis-Menten constants were as follows: KII: Km 2.55 +/- 0.22 mM, Vmax 0.357 +/- 0.017 mumol/min/mL; CN1: Km 6.93 +/- 0.32 mM, Vmax 0.748 +/- 0.019 mumol/min/mL; and CN2: Km 35.8 +/- 1.52 mM, Vmax 13.11 +/- 0.40 mumol/min/mL. EDTA and O-phenanthroline inhibited the three enzyme assays at the same Ki, whereas captopril and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA), allowed for the demonstration of the specificity of each assay. Furthermore, Ki values of MERGETPA against both CN1 (4.75 microM) and CN2 (2.36 microM) activities do not support the hypothesis that KI activity may be accounted for by the presence of isoenzymes in rat plasma.
Barabé
J
J
Département de Pharmacologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Huberdeau
D
D
D000818:Animals; D002216:Captopril / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004492:Edetic Acid / Q000494:pharmacology; D006626:Hippurates / Q000032:analysis; D007700:Kinetics; D001121:Lysine Carboxypeptidase / Q000037:antagonists & inhibitors / Q000097:blood*; D008838:Microcomputers; D009842:Oligopeptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000097:blood*; D010618:Phenanthrolines / Q000494:pharmacology; D051381:Rats
D006626:Hippurates; D009842:Oligopeptides; D010618:Phenanthrolines; C010980:hippuryl-histidyl-leucine; D004492:Edetic Acid; D002216:Captopril; D007703:Peptidyl-Dipeptidase A; D001121:Lysine Carboxypeptidase; C030514:hippuric acid