Characterization of chicken lung angiotensin I-converting enzyme

• DOI: 10.1677/joe.0.1320261
• Publication Date: 2008-12-11T23:11:46Z
• Authors: M. J. Polanco; J. L. Miguel; M. T. Agapito; J. M. Recio
• Keywords: Zinc; Carbohydrates; Animals; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Peptidyl-Dipeptidase A; Chickens; Lung; Chromatography, Affinity

ABSTRACT Angiotensin I(AI)-converting enzyme (ACE) (EC 3.4.15.1) was solubilized from the membrane fraction of chicken lung using trypsin and nonidet P40 extraction, and then purified to homogeneity by captopril affinity chromatography. Comparison of trypsin-extracted and detergent-solubilized membrane-bound converting enzyme by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane-binding sequence contributed to a large extent to the size and charge of the enzyme. Both forms of the enzyme were glycoproteins but they differed in the glucidic content; 4·5% by weight of the enzyme in the trypsin-extracted ACE and 15% by weight of the enzyme in the detergent-solubilized ACE. In both cases hexoses were the most abundant residues. Both forms of the enzyme were found to contain 1 g-atom zinc/mol enzyme. The purified enzymes did not only split Hip-His-Leu but also AI and bradykinin. The Michaelis constant (Km) and maximum velocity (Vmax) values of the trypsin-extracted ACE for Hip-His-Leu were 52 × 10−5 mol/l and 15·36 nmol/min respectively, and for AI they were 7·8 × 10−5 mol/l and 0·45 nmol/min respectively. The Km and Vmax values of the detergent-solubilized ACE for Hip-His-Leu were 32 × 10−5 mol/l and 11·75 nmol/min respectively, and for AI they were 6·5 × 10−5 mol/l and 0·97 nmol/min. Both forms of the enzyme required the same chloride ion concentration (0·3 mol/l) for the optimum enzyme activity and showed the same pH optimum (8·5) with Hip-His-Leu as substrate. Both were inhibited by EDTA in a similar manner. In addition, they were inhibited by the peptides AII and His-Leu, but captopril was the most potent inhibitor with inhibition constant values of 1·6 nmol/l and 2·0 nmol/l for the trypsin-extracted and the detergent-solubilized ACE respectively. Journal of Endocrinology (1992) 132, 261–268