The active site of endopeptidase-24.11: substrate and inhibitor studies.

Quite a few years ago, while studying the inactivation of bradykinin by kidney extracts, an enzyme in a particulate (microsomal) fraction of rat kidney was found to release the C-terminal dipeptide (Phe-Arg) of bradykinin (Erdos & Yang, 1967). This enzyme, named kininase 11, was solubilized with deoxycholate and partially purified by gel filtration. It was subsequently also isolated from human plasma (Yang & Erdos, 1967). Prior to that an ACE, was found in horse plasma (Skeggs et al., 1954, 1956). ACE was shown to be a peptidyl dipeptidase (peptidyl dipeptidase A; EC 3.4.15.1) identical with kininase I1 after testing it with a variety of peptide substrates (Yang et al., 1970, 1971). Vane, Ferreira and their colleagues originally pointed out the importance of the lung in the metabolism of circulating peptides (Ferreira & Vane, 1967; Ng & Vane, 1967, 1968; Vane, 1969). The lung is a rich source of ACE and the enzyme is bound to the plasma-membrane of vascular endothelial cells (Ryan et al., 1976). Endothelial cells of extrapulmonary blood vessels also contain ACE (Kreye & Gross, 1971; Aiken & Vane, 1972; Rabito et al., 1972). Many other organs contain high concentrations of ACE where it is concentrated mainly in epithelial cells, e.g. the microvilli of the gut (Ward et al., 1980), kidney (Hall et al., 1976), placenta (Johnson et al., 1984) and choroid plexus (Igic et al., 1977; Rix et al., 1981). The enzyme was also detected in various parts of rat and human brain and in the pituitary gland (Yang & Neff, 1972; Poth et al., 1975). To localize ACE in tissues and organs by immunohistochemical techniques, the enzyme had to be purified in order to elicit a specific antiserum. The purification of ACE from human tissues was a more complex problem than the purification from organs of hog (Oshima et al., 1974; Nakajima et al., 1973) or rabbit (Cushman & Cheung, 1972; Das & Soffer, 1975), since the human enzyme appears to be more hydrophobic than the animal variety. This leads to significant losses of activity during the purification procedures. By employing trypsin to solubilize ACE from a membrane fraction (Nishimura et al., 1977; Stewart et al., 1981), and the technique of ‘reverse immunoadsorption’ (Weare et al., 1982), the purification procedure was greatly simplified. ACE in human kidney is 5-6 times more concentrated per weight than in the lung and it is bound to the brush border in the proximal tubules (Weare et al., 1982). Presumably, ACE is a transmembrane peptidase with a hydrophobic anchor peptide inserted into the lipid bilayer of the plasma membrane (Erdos & Gafford, 1983). The difference in M , between the membrane-bound enzyme and the enzyme solubilized with trypsin was shown with ‘Western blotting’. ACE extracted from the renal membrane with detergent had a slightly higher M , value than the enzyme purified from kidney after trypsin treatment (Erdos & Gafford,