Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid.
Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters-ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 degrees C for 30 min-the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.
Lam
Le Hoang
le H
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan.
Shimamura
Tomoko
T
Sakaguchi
Ken
K
Noguchi
Katsuya
K
Ishiyama
Munetaka
M
Fujimura
Yume
Y
Ukeda
Hiroyuki
H
D020155:3-Hydroxybutyric Acid / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry* / Q000145:classification*; D000818:Animals; D001671:Biochemistry / Q000379:methods; D002138:Calibration; D002216:Captopril / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019612:Fagopyrum / Q000737:chemistry; D005240:Feasibility Studies; D005504:Food Analysis / Q000379:methods; D005562:Formazans / Q000737:chemistry; D005737:Garlic / Q000737:chemistry; D006626:Hippurates / Q000737:chemistry; D006868:Hydrolysis; D006885:Hydroxybutyrates; D007202:Indicators and Reagents; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D009842:Oligopeptides / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D011817:Rabbits; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013379:Substrate Specificity
C522037:3-hydroxybutyrylglycyl-glycyl-glycine; D000806:Angiotensin-Converting Enzyme Inhibitors; D005562:Formazans; D006626:Hippurates; D006885:Hydroxybutyrates; D007202:Indicators and Reagents; D009842:Oligopeptides; D002216:Captopril; C030514:hippuric acid; D020155:3-Hydroxybutyric Acid