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The renin-angiotensin-aldosterone system (RAAS) is a key regulator of systemic blood pressure and renal function and a key player in renal and cardiovascular disease. However, its (patho)physiological roles and its architecture are more complex than initially anticipated. Novel RAAS components that may add to our understanding have been discovered in recent years. In particular, the human homologue of ACE (ACE2) has added a higher level of complexity to the RAAS. In a short period of time, ACE2 has been cloned, purified, knocked-out, knocked-in; inhibitors have been developed; its 3D structure determined; and new functions have been identified. ACE2 is now implicated in cardiovascular and renal (patho)physiology, diabetes, pregnancy, lung disease and, remarkably, ACE2 serves as a receptor for SARS and NL63 coronaviruses. This review covers available information on the genetic, structural and functional properties of ACE2. Its role in a variety of (patho)physiological conditions and therapeutic options of modulation are discussed., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

In contrast to the relatively ubiquitous angiotensin-converting enzyme (ACE), expression of the mammalian ACE homologue, ACE2, was initially described in the heart, kidney and testis. ACE2 is a type I integral membrane protein with its active site domain exposed to the extracellular surface of endothelial cells and the renal tubular epithelium. Here ACE2 is poised to metabolise circulating peptides which may include angiotensin II, a potent vasoconstrictor and the product of angiotensin I cleavage by ACE. To this end, ACE2 may counterbalance the effects of ACE within the renin-angiotensin system (RAS). Indeed, ACE2 has been implicated in the regulation of heart and renal function where it is proposed to control the levels of angiotensin II relative to its hypotensive metabolite, angiotensin-(1-7). The recent solution of the structure of ACE2, and ACE, has provided new insight into the substrate and inhibitor profiles of these two key regulators of the RAS. As the complexity of this crucial pathway is unravelled, there is a growing interest in the therapeutic potential of agents that modulate the activity of ACE2.

Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.

Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.

A novel human zinc metalloprotease that has considerable homology to human angiotensin-converting enzyme (ACE) (40% identity and 61% similarity) has been identified. This metalloprotease (angiotensin-converting enzyme homolog (ACEH)) contains a single HEXXH zinc-binding domain and conserves other critical residues typical of the ACE family. The predicted protein sequence consists of 805 amino acids, including a potential 17-amino acid N-terminal signal peptide sequence and a putative C-terminal membrane anchor. Expression in Chinese hamster ovary cells of a soluble, truncated form of ACEH, lacking the transmembrane and cytosolic domains, produces a glycoprotein of 120 kDa, which is able to cleave angiotensin I and angiotensin II but not bradykinin or Hip-His-Leu. In the hydrolysis of the angiotensins, ACEH functions exclusively as a carboxypeptidase. ACEH activity is inhibited by EDTA but not by classical ACE inhibitors such as captopril, lisinopril, or enalaprilat. Identification of the genomic sequence of ACEH has shown that the ACEH gene contains 18 exons, of which several have considerable size similarity with the first 17 exons of human ACE. The gene maps to chromosomal location Xp22. Northern blotting analysis has shown that the ACEH mRNA transcript is approximately 3. 4 kilobase pairs and is most highly expressed in testis, kidney, and heart. This is the first report of a mammalian homolog of ACE and has implications for our understanding of cardiovascular and renal function.

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Angiotensin-converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein are two examples of membrane-bound proteins that are released in a soluble form by a post-translational proteolytic cleavage event involving a secretase. Site-specific antibodies and matrix-assisted laser desorption ionization-time-of-flight ('MALDI-TOF') MS have been used to map the secretase cleavage site in somatic ACE to Arg-1203/Ser-1204, 24 residues proximal to the membrane-anchoring domain. Trypsin, which can solubilize ACE from the membrane, cleaves the protein at the same site. The use of structurally related hydroxamic acid-based zinc metalloproteinase inhibitors indicate that tumour necrosis factor-alpha convertase, a member of the ADAMs ('a disintegrin and metalloproteinase') family of proteins, is not involved in the proteolytic release of ACE, or in the constitutive or regulated alpha-secretase release of the amyloid precursor protein from a human neuronal cell line.

Angiotensin converting enzyme (ACE) and the Alzheimer's amyloid precursor protein are cleaved from the membrane by zinc metalloproteinases termed ACE secretase and alpha-secretase, respectively. Tumour necrosis factor-alpha (TNF-alpha) convertase (ADAM 17) is a recently identified member of the adamalysin family of mammalian zinc metalloproteinases that is involved in the production of TNF-alpha and possibly in the cleavage of other membrane proteins. Using two different cell-free assays we were unable to detect significant cleavage and secretion of ACE by TNF-alpha convertase. In addition, there was a different effect of three hydroxamic acid-based inhibitors (batimastat, compound 1 and compound 4) towards TNF-alpha convertase as compared to ACE secretase and alpha-secretase. Thus TNF-alpha convertase would appear to be distinct from, but possibly related to, the secretases that cleave ACE and the amyloid precursor protein.

The 4 kDa beta-amyloid peptide that forms the amyloid fibrils in the brain parenchyma of Alzheimer's disease patients is derived from the larger integral membrane protein, the amyloid precursor protein. In the nonamyloidogenic pathway, alpha-secretase cleaves the amyloid precursor protein within the beta-amyloid domain, releasing an extracellular portion and thereby preventing deposition of the intact amyloidogenic peptide. The release of the amyloid precursor protein from both SH-SY5Y and IMR-32 neuronal cells by alpha-secretase was blocked by batimastat and other related synthetic hydroxamic acid-based zinc metalloprotease inhibitors, but not by the structurally unrelated zinc metalloprotease inhibitors enalaprilat and phosphoramidon. Batimastat inhibited the release of the amyloid precursor protein from both cell lines with an I50 value of 3 microM. Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat toward alpha-secretase. In the SH-SY5Y cells, both the basal and the carbachol-stimulated release of the amyloid precursor protein were blocked by batimastat. In contrast, neither the level of full-length amyloid precursor protein nor its cleavage by beta-secretase were inhibited by any of the zinc metalloprotease inhibitors examined. In transfected IMR-32 cells, the release of both the amyloid precursor protein and angiotensin converting enzyme was inhibited by batimastat, marimastat, and BB2116 with I50 values in the low micromolar range, while batimastat and BB2116 inhibited the release of both proteins from HUVECs. The profile of inhibition of alpha-secretase by batimastat and structurally related compounds is identical with that observed with the angiotensin converting enzyme secretase suggesting that the two are closely related zinc metalloproteases.

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.

Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.

The molecular forms of angiotensin converting enzyme (ACE; EC 3.4.15.1) in preparations of pig brain cortical microvessels and striatal synaptosomal membranes have been identified by immunoelectrophoretic blot analysis. The cortical microvessels contained only the endothelial form of the enzyme, Mr 180,000, which comigrated with pig kidney ACE on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, the synaptosomal membranes contained only a smaller form of ACE, Mr 170,000, which represents the neuronal form of the enzyme. No significant differences in inhibitor sensitivity or substrate specificity were detected between the two forms of ACE. In particular, neurokinin A was resistant to hydrolysis by either microvessel or synaptosomal membrane ACE, and the pattern of hydrolysis of substance P by the two preparations was identical.

1. The two isozymes of human angiotensin converting enzyme (ACE; EC 3.4.15.1) have recently been cloned and sequenced. 2. The larger, endothelial isozyme has two highly similar internal domains each bearing a putative catalytic site. In contrast the smaller, testicular isozyme has a single catalytic site corresponding to the C-terminal domain of endothelial ACE and represents the ancestral, non-duplicated form of the gene. 3. Both isozymes are anchored in the plasma membrane by a single hydrophobic transmembrane polypeptide located near the C-terminus, and both are extensively N-glycosylated. 4. The testicular isozyme may also be O-glycosylated. 5. The soluble form of ACE in plasma, seminal fluid and other body fluids appears to be derived from the membrane-bound endothelial isozyme by a post-translational modification. 6. ACE has a complex substrate specificity with peptidyl tripeptidase or endopeptidase action on certain peptides, as well as the classical peptidyl dipeptidase activity. 7. Numerous potent inhibitors of the enzyme have been developed and used successfully in the treatment of hypertension, but some of the observed side effects may be due to inhibition of other zinc metalloenzymes. 8. Both endothelial and testicular ACE are highly conserved between species, indicative of the essential role(s) of the enzyme in blood pressure regulation and other physiological processes.

Peptidyl-dipeptidase A (angiotensin converting enzyme; ACE, EC 3.4.15.1), has been purified from pig kidney and striatum by affinity chromatography employing the selective inhibitor lisinopril as ligand. The inclusion of a 2.8 nm spacer arm improved the yield of the enzyme compared with the 1.4 nm spacer arm described in previous work. Two forms of striatal ACE (Mr 180,000 and 170,000), but only a single form of kidney ACE (Mr 180,000), were isolated by this procedure. Both forms of striatal ACE were recognized by a polyclonal antibody to kidney ACE. No significant differences in substrate specificity or inhibitor sensitivity between kidney and striatal ACE could be detected. In particular, the amidated neuropeptide, substance P, was hydrolysed identically by both preparations and no significant hydrolysis of the related tachykinin peptides neurokinin A and neurokinin B could be detected. After chemical or enzymic deglycosylation, kidney and both forms of striatal ACE migrated identically on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 150,000. We suggest that the two detectable forms of ACE in pig brain are not isoenzymes but are the result of differential glycosylation in different cell types in the brain. It appears that ACE, unlike endopeptidase-24.11, does not have the general capacity to hydrolyse and inactivate the tachykinin peptides at a significant rate in brain.

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.

Endopeptidase-24.11 (sometimes referred to as 'enkephalinase') is a key cell-surface enzyme in the metabolism of neuropeptides. A previous immunohistochemical study mapped the enzyme in pig brain and indicated a striosomal ordering of the enzyme within the striatum. This point has now been confirmed by staining adjacent sections for acetylcholinesterase (by histochemistry) and endopeptidase-24.11 (by an immunoperoxidase method). While there were some general similarities in the mapping of these two hydrolases, e.g. in the caudate-putamen, globus pallidus, olfactory tubercle, substantia nigra and striatonigral tract, there were differences in intensity and in the microscopic distribution, e.g. as in striosomes for which acetylcholinesterase was diminished. Two other membrane peptidases, peptidyl dipeptidase A ('angiotensin converting enzyme') and aminopeptidase N, were also mapped by the same immunohistochemical method. Peptidyl dipeptidase A had some similarities with endopeptidase-24.11, e.g. in its concentration within the striatal nuclei, but clear differences were also apparent, in particular the absence of staining of the former in the globus pallidus and olfactory tubercle. Immunostaining for aminopeptidase N, in contrast to the other peptidases, was observed as a diffuse staining throughout the gray matter. At the microscopic level, two important differences were that staining for aminopeptidase N and peptidyl dipeptidase A was very intense throughout the vasculature of the brain and that striatal efferent bundles of unmyelinated fibres staining positively for endopeptidase-24.11 were depleted of the other two peptidases. All three peptidases were identified in the pia mater. Thus, endopeptidase-24.11, unlike peptidyl dipeptidase A and aminopeptidase N, is a marker for a set of striatal efferent fibres in pig brain.

The major site of hydrolysis was the Gly8-Leu9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C-terminal tripeptide Gly-Leu-MetNH2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase-24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin-sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase-24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2).

Angiotensin converting enzyme from pig kidney was isolated by affinity chromatography after solubilization from the membrane by one of four different procedures. Solubilization with Triton X-100, trypsin or by an endogenous activity in microvillar membranes all generated hydrophilic forms of the enzyme as assessed by phase separation in Triton X-114 and failure to incorporate into liposomes. Only when solubilization and purification was effected by Triton X-100 in the presence of EDTA (10 mM) could an amphipathic form of the enzyme (membrane- or m-form) be generated. The m-form of angiotensin converting enzyme (ACE) appeared slightly larger (Mr approx. 180,000) than the hydrophilic forms (Mr approx. 175,000) after SDS/polyacrylamide-gel electrophoresis, and the m-form incorporated into liposomes, consistent with retention of the membrane anchor. The m-form of ACE showed an N-terminal sequence identical with that of preparations of enzyme isolated after solubilization with detergent alone (d-form), with trypsin (t-form) or by the endogenous mechanism (e-form). These data imply that ACE is anchored to the plasma membrane via its C-terminus, in contrast with the N-terminal anchorage of endopeptidase-24.11. No release of ACE from the membrane could be detected with a variety of phospholipases, including bacterial phosphatidylinositol-specific phospholipases C, although an endogenous EDTA-sensitive membrane-associated hydrolase was capable of releasing a soluble, hydrophilic, form of the enzyme.

The electric organ of Torpedo marmorata contains a membrane-bound, captopril-sensitive metallopeptidase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chromatography using the selective inhibitor lisinopril immobilised to Sepharose via a 28-A spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C-terminus of a variety of peptide substrates, including angiotensin I, bradykinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)-His-Leu. The hydrolysis of BzGly-His-Leu was activated by Cl-. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuropeptide substance P. Hydrolysis of substance P occurred primarily at the Phe8-Gly9 bond with release of the C-terminal tripeptide, Gly-Leu-MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr = 190,000) than the pig kidney enzyme (Mr = 180,000) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.