Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.     

Characterisation of Neutral Endopeptidase 3.4.24.11 (NEP) in the Kidney: Comparison Between Normotensive,Genetically Hypertensive and Experimentally Hypertensive Rats

Abstract

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY). DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters,: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280μM), (3) IC₅₀ of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH8.0), (5) heat inactivation profiles (half-life 20min at 65°C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.     

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.     

A peptidyl dipeptidase-4 from Pseudomonas maltophilia: purification and properties

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.     

Effect of neutral endopeptidase inhibition of f-Met-Leu-Phe-induced bronchoconstriction in the rabbit

Inhalation of f-Met-Leu-Phe (FMLP) produces dose-dependent increases in pulmonary resistance (RL) in rabbits. We hypothesized that inhibition of neutral endopeptidase (NEP), which has high affinity for FMLP, would augment the response to FMLP inhalation. We found the increase in RL above baseline in response to FMLP to be reduced from 56 +/- 18 to 8 +/- 10% (P less than 0.01) by phosphoramidon (1 mg/kg) and to 15 +/- 6% (P less than 0.02) by thiorphan (3 mg/kg). The geometric mean dose of FMLP producing a 20% rise in RL (PC20RL FMLP) was increased by phosphoramidon from 1.1 to 4.5 mg/ml (P less than 0.05). Enkephalins, which are also NEP substrates, modulate cholinergic neurotransmission in the airway. Inhibition of the FMLP response by phosphoramidon was reversed by coadministration of naloxone (0.1 mg/kg); after atropine (2 mg/kg) the change in RL in response to FMLP was reduced to 7 +/- 4% (P less than 0.01), whereas morphine (0.15 mg/kg) increased PC20RL FMLP to 5.1 mg/ml (P less than 0.05). FMLP-induced bronchoconstriction in the rabbit is vagally mediated, and reduced responses after NEP inhibition may reflect modulation of cholinergic bronchoconstriction by enkephalins. Changes in airway NEP activity may influence the activity of a wide range of its substrates, of which some are bronchoconstrictors and others bronchodilators.     

Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors.

Neutral endopeptidase (EC 3.4.24.11) from pig kidney hydrolyses [125I]iodo-insulin B-chain and leucine-enkephalin. Both activities were equally sensitive to inhibition by phosphoramidon [N-(alpha-L-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan] and thiorphan [N-(DL-2-benzyl-3-mercaptopropionyl)glycine]. Thermolysin hydrolysis of insulin B-chain was also sensitive to both inhibitors. The hydrolysis of the Gly3-Phe4 bond of Leu-enkephalin by synaptic membranes prepared from pig brain was partially inhibited by phosphoramidon and thiorphan. Synaptic membranes appear to contain another endopeptidase activity that is insensitive to these reagents. These observations suggest that enzymes similar to the kidney endopeptidase may play a general role in neuropeptide metabolism.     

Characterisation of neutral endopeptidase 3.4.24.11 (NEP) in the kidney: comparison between normotensive, genetically hypertensive and experimentally hypertensive rats.

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY), DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280 microM), (3) IC50 of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH 8.0), (5) heat inactivation profiles (half-life 20 min at 65 degrees C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.     

A three-dimensional model of endothelin-converting enzyme (ECE) based on the X-ray structure of neutral endopeptidase 24.11 (NEP).

Endothelin-converting enzyme 1 (ECE-1, EC 3.4.24.71) is a zinc-dependent type II mammalian membrane protein comprising the active site in the ectodomain. It exists in multiple splice variants that all catalyze the last and rate-limiting step in the activation of preproendothelin to the highly potent vasoconstrictor endothelin. There is high interest in finding small and potent inhibitors for this enzyme that could be used in numerous indications, e.g. hypertension. Since there is no structural information available for this important enzyme, we built a model of the complete ectodomain using the recently solved structure of human NEP as template. The naturally derived metalloproteinase inhibitor phosphoramidon was docked in the active site of this model and comparisons with the respective NEP complex were made.     

Structure-based design of dipeptide derivatives for the human neutral endopeptidase

Neutral endopeptidase (NEP) plays a key role in the metabolic inactivation of various bioactive peptides such as atrial natriuretic peptide (ANP), endothelins, and enkephalins. Furthermore, NEP is known to work as elastase in skin fibroblast. Therefore, effective inhibitors of NEP offer significant therapeutic interest as antihypertensives, analgesics, and skin anti-aging agents. Recently, the X-ray crystal structure of human NEP complexed with phosphoramidon has been reported and provided insights into the active site specificity of NEP. Here, we designed new inhibitors by using in silico molecular modeling and synthesized them by short steps. Expectedly, we found highly effective inhibitors with sub-nanomolar levels of IC(50) values. These results indicate that our structure-based molecular designing program is useful for obtaining novel NEP inhibitors. Furthermore, these inhibitors may be attractive leads for the generation of new pharmaceuticals for NEP-related diseases.     

The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function

Neprilysin (NEP), a thermolysin-like zinc metalloendopeptidase, plays an important role in turning off peptide signalling events at the cell surface. It is involved in the metabolism of a number of regulatory peptides of the mammalian nervous, cardiovascular, inflammatory and immune systems. Examples include enkephalins, tachykinins, natriuretic and chemotactic peptides. NEP is an integral plasma membrane ectopeptidase of the M13 family of zinc peptidases. Other related mammalian NEP-like enzymes include the endothelin-converting enzymes (ECE-1 and ECE-2), KELL and PEX. A number of novel mammalian homologues of NEP have also recently been described. NEP family members are potential therapeutic targets, for example in cardiovascular and inflammatory disorders, and potent and selective inhibitors such as phosphoramidon have contributed to understanding enzyme function. Inhibitor design should be facilitated by the recent three-dimensional structural solution of the NEP-phosphoramidon complex. For several of the family members, however, a well-defined physiological function or substrate is lacking. Knowledge of the complete genomes of Caenorhabditis elegans and Drosophila melanogaster allows the full complement of NEP-like activities to be analysed in a single organism. These model organisms also provide convenient systems for examining cell-specific expression, developmental and functional roles of this peptidase family, and reveal the power of functional genomics.     

The role of neutral endopeptidase in caerulein-induced acute pancreatitis

Substance P (SP) is well known to promote inflammation in acute pancreatitis (AP) by interacting with neurokinin-1 receptor. However, mechanisms that terminate SP-mediated responses are unclear. Neutral endopeptidase (NEP) is a cell-surface enzyme that degrades SP in the extracellular fluid. In this study, we examined the expression and the role of NEP in caerulein-induced AP. Male BALB/c mice (20-25 g) subjected to 3-10 hourly injections of caerulein (50 μg/kg) exhibited reduced NEP activity and protein expression in the pancreas and lungs. Additionally, caerulein (10(-7) M) also downregulated NEP activity and mRNA expression in isolated pancreatic acinar cells. The role of NEP in AP was examined in two opposite ways: inhibition of NEP (phosphoramidon [5 mg/kg] or thiorphan [10 mg/kg]) followed by 6 hourly caerulein injections) or supplementation with exogenous NEP (10 hourly caerulein injections, treatment of recombinant mouse NEP [1 mg/kg] during second caerulein injection). Inhibition of NEP raised SP levels and exacerbated inflammatory conditions in mice. Meanwhile, the severity of AP, determined by histological examination, tissue water content, myeloperoxidase activity, and plasma amylase activity, was markedly better in mice that received exogenous NEP treatment. Our results suggest that NEP is anti-inflammatory in caerulein-induced AP. Acute inhibition of NEP contributes to increased SP levels in caerulein-induced AP, which leads to augmented inflammatory responses in the pancreas and associated lung injury.     

The study of substrate specificity of a new peptide substrate of endothelin-converting enzyme

A new hexapeptide CMC-Ala-Gly-Gly-Trp-Phe-Arg was used as a substrate for assay of endothelin-converting (ECE; EC 3.4.24.71) and angiotensin-converting (ACE; EC 3.4.15.1) enzymes and of neutral endopeptidase (NEP; EC 3.4.24.11). The specific inhibitors lisinopril (for ACE) and thiorphan (for NEP) were used for discrimination between activities of these enzymes.     

Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.     

Characterization of phosphoramidon-sensitive metalloproteinases with endothelin-converting enzyme activity in porcine lung membrane

Endothelin-1 (ET-1), a 21 amino-acid potent vasoconstrictor peptide, is produced from the biologically inactive intermediate big ET-1 via an endoproteolytic cleavage between Trp-21 and Val-22 by endothelin converting enzyme (ECE). cDNA sequence analysis predicts that the two other members of the endothelin family, ET-2 and ET-3, are also generated from the corresponding intermediates called big ET-2 and big ET-3, respectively. The metalloproteinase inhibitor phosphoramidon inhibited the conversion of big ET-1 into mature ET-1 both in vivo and in cultured endothelial cells, suggesting that ECE may be a neutral metalloproteinase. In this study, we solubilized and partially purified ECE from the membrane fraction of porcine lung. Using gel filtration chromatography, we separated two distinct ECE activities, designated M1 (apparent molecular mass approx. 300 kDa) and M2 (approx. 65 kDa). Optimum pH for the cleavage of big ET-1 by M1 and M2 was 7.0 and 7.5, respectively. M1 efficiently converted human big ET-1(1–38) to ET-1, but not human big ET-2(1–37) or human big ET-3(1–41)-amide. In contrast, M2 converted both big ET-1 and big ET-2, but not big ET-3. M1 was inhibited by phosphoramidon (IC₅₀ approx. 1 μM) but not by thiorphan or bacitracin. In contrast, M2 was inhibited by much lower concentrations of phosphoramidon (IC₅₀ approx. 0.3 nM), as well as by thiorphan and bacitracin. ECE activity in M1 was able to bind to a concanavalin A-agarose column and was eluted by α-methyl-d-glucoside, indicating that the ECE is glycosylated. From these results, M1 and M2 from the porcine lung membrane are similar to the candidate of ECE in endothelial cells and neutral endopeptidase in kidney (EC 3.4.24.11), respectively. Taken in conjunction with the previous finding that neither thiorphan nor bacitracin affected the conversion of endogenously synthesized big ET-1 in cultured endothelial cells, we conclude that physiologically relevant ECE found in the endothelial cells is more similar to M1 than to M2.     

Neutral endopeptidase modulates neurotensin-induced airway contraction

To determine the role of endogenous neutral endopeptidase (NEP) (also called enkephalinase, EC 3.4.24.11) in regulating neurotensin-induced airway contraction, we used phosphoramidon, a specific NEP inhibitor, in the guinea pig. In studies in vitro, neurotensin and the COOH-terminal fragment neurotensin-(8-13) contracted strips of bronchial smooth muscle in a concentration-dependent fashion (P less than 0.001). In contrast, the NH2-terminal fragment neurotensin-(1-11) and the COOH-terminal fragment neurotensin-(12-13), the main fragments of neurotensin hydrolysis by NEP, had no effect. Phosphoramidon (10(-5) M) did not change resting tension but shifted the concentration-response curves to neurotensin to lower concentrations (P less than 0.001), whereas inhibitors of kininase II, aminopeptidases, serine proteases, and carboxypeptidase N were without effect. Removing the epithelium increased the contractile response to neurotensin (P less than 0.001), and phosphoramidon further increased the response to neurotensin in these tissues (P less than 0.001). Similar results were obtained in studies in vivo using aerosolized neurotensin and phosphoramidon. These results suggest that endogenous NEP in the airways modulates the effects of neurotensin on airway smooth muscle contraction by inactivating the peptide.     

A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11)

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.     

The production and characterization of a monoclonal antibody specific for the 94,000 dalton enkephalin-degrading peptidase from rabbit kidney brush border

We have prepared a monoclonal antibody specific for a major 94,000 dalton protein from the brush border membrane of rabbit kidney cortex. The monoclonal antibody was used for the immunoaffinity purification of this protein after solubilization of brush border membranes with octylglucoside. The 94,000 dalton protein is a peptidase capable of cleaving the Gly3-Phe4 bond of methionine-enkephalin. Identification of this peptidase as a previously described 94,000 dalton enkephalinase of kidney cortex was confirmed by its sensitivity to EDTA and inhibitors such as thiorphan and phosphoramidon.     

Angiotensin converting enzyme has an inhibitory role in CGRP metabolism in human skin

The neutral endopeptidase (NEP) is important for calcitonin gene related peptide (CGRP) degradation, while the role of angiotensin converting enzyme (ACE) remains unclear. By using dermal microdialysis we explored the effect of phosphoramidon (NEP blocker), captopril (ACE blocker) and a mixture of both drugs on the intensity of electrically-induced CGRP-mediated neurogenic flare. The results reveal that phosphoramidon elevated flare intensity, but that this was not further increased by adding captopril. In contrast, neurogenic flare was decreased when the drug mixture was applied in compared to NEP only. Electrically released CGRP levels could be measured directly in perfusates containing phosphoramidon and the mixture. Again, CGRP levels were elevated in phosphoramidon treated sites, and significantly reduced upon adding captopril. These findings suggest that NEP and ACE do not have additive effects regarding neuropeptide degradation. In contrast, inhibition of ACE seems to augment CGRP catabolism.     

Identification of endothelin converting enzyme in bovine lung membranes using a new fluorogenic substrate.

An enzyme partially purified from bovine lung membranes appears to be endothelin converting enzyme (ECE). This enzyme specifically cleaves big endothelin-1 (big ET-1) at the proper site, between Trp21 and Val22, with maximum activity at pH 7.5 and with a Km of roughly 3 microM, to produce endothelin-1 (ET-1) and C-terminal peptide (CTP). This same enzyme hydrolyzes the fluorogenic substrate succinyl-Ile-Ile-Trp-methylcoumarinamide to release the highly fluorescent 7-amino-4-methylcoumarin. The peptide derivative has the same amino acid sequence as big ET-1 and is a good substrate with a Km of about 27 microM. This enzyme is a metalloproteinase. It is not inhibited by five common proteinase inhibitors (pepstatin A, PMSF, NEM, E-64 and thiorphan) but it is inhibited by phosphoramidon and chelating compounds. The apoenzyme is restored to nearly full activity by a zinc-EDTA buffer with pZn = 13.