Hydrolysis of Peptide Hormones by Endothelin-converting Enzyme-1
A COMPARISON WITH NEPRILYSIN*

Gary D. JohnsonDagger , Tracy Stevenson§, and Kyunghye AhnDagger

From the Dagger  Department of Biochemistry and § Department of Chemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105

    ABSTRACT
Top
Abstract
Introduction
References

Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins.

    INTRODUCTION
Top
Abstract
Introduction
References

Endothelins (ETs)1 are potent vasoconstrictive peptides of 21 amino acids produced by vascular endothelial cells (1). Three ET isoforms, ET-1, ET-2, and ET-3, encoded by distinct genes, are known to exist in humans (2). Endothelins are involved in the regulation of vascular tone and may also play roles in various cardiovascular and renal diseases (3). ETs are also required during embryonic development for the intercellular signaling necessary for the proper development of neural crest-derived tissues (4). The final step in the biosynthesis of the endothelins is the conversion of 38-41 residue precursors (big ETs) to the active hormones via the cleavage of a Trp21-Val/Ile22 bond by endothelin-converting enzymes (ECEs (5)). ECE-1 has been purified from vascular endothelium, endothelial cell lines, and lung microsomes (6-8). ECE-1 (EC 3.4.24.71) is a Type II integral membrane protein expressed by endothelial cells in tissues such as aorta, lung, ovary, and testis. It has also been reported to be expressed by endocrine cells such as adrenal chromaffin cells and pancreatic beta  cells (9). Targeted disruption of the ECE-1 gene has shown that ECE-1 is the physiologically relevant activating enzyme for both ET-1 and ET-3 in vivo (10).

Molecular cloning of mammalian ECE-1 cDNAs has demonstrated the existence of three mRNAs transcribed from a single gene (11, 12). The proteins encoded by these RNAs have identical catalytic domains but differ only in their NH2-terminal amino acid sequence. Two of the ECE-1 isoforms are expressed on the cell surface; the other is localized in the trans-Golgi network (12) An additional isoform (ECE-2), 59% identical in amino acid sequence to ECE-1, that appears to be expressed in the trans-Golgi network has also been identified (13). The endothelin-converting enzymes belong to a family of metallopeptidases including neprilysin (neutral endopeptidase 24.11, NEP) and Kell, an antigen expressed on the surface of erythrocytes (14). These proteins have the greatest amount of sequence identity in their COOH-terminal regions, especially in residues involved in zinc binding and catalysis, indicating a similar structure and catalytic mechanism for all. NEP is the most extensively characterized enzyme of this group (15). NEP cleaves a variety of biologically active peptides, usually at the amino side of hydrophobic residues. Studies using big ETs and peptides derived from endothelins have led to the conclusions that hydrolysis of substrates by ECE-1 may be highly dependent on substrate conformation and that the enzyme may have a narrow substrate specificity (8, 16). However, previous studies of ECE-1 peptidase activity have been hampered by the limited availability of the pure enzyme and have rarely used peptides other than big endothelins as substrates. A more thorough examination of ECE-1 activity and substrate specificity is required to better understand its mechanism of action and to identify additional in vivo substrates. As an initial step in the systematic examination of ECE-1 substrate specificity, we have purified recombinant soluble ECE-1 (solECE-1) to homogeneity and examined its activity against a number of biologically active peptides. The results indicate that ECE-1 can hydrolyze a broad spectrum of peptide substrates with a specificity similar to that of NEP.

    EXPERIMENTAL PROCEDURES

Expression and Purification of solECE-1-- The human ECE-1a cDNA was modified so that the extracellular domain (amino acids 78-758) was fused in-frame to a DNA sequence encoding the signal sequence of human alkaline phosphatase. The modified cDNA was subcloned into the mammalian expression vector pSG5 (Stratagene, La Jolla, CA) with protein expression driven by a SV40 promoter. A stably transfected Chinese hamster ovary K1 cell line harboring the resultant plasmid secreted solECE-1 into the culture medium. The purification of solECE-1 from this conditioned medium has been recently described in detail (32). Briefly, the purification involved successive chromatographic steps utilizing the binding of solECE-1 to DEAE-agarose, wheat germ agglutinin-agarose, and alkyl-Superose resins. The purification of solECE-1 was monitored by assaying the conversion of human big ET-1 to ET-1 using an enzyme-linked immunosorbent assay kit for the quantitation of ET-1 (Amersham Pharmacia Biotech). The solECE-1 was judged to be homogeneous when analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining.

Materials-- Arg-Pro-Pro-Gly-Phe-Ser-Pro (bradykinin (1-7)) and Asp-Arg-Val-Tyr (angiotensin I (1-4)) were purchased from American Peptide Co. (Sunnyvale, CA). Arg-Pro-Lys-Pro-Gln-Gln (substance P (1-6)), pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro (neurotensin (1-10)), and Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (neurotensin (3-13)) were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). Oxidized insulin B chain, dansyl-D-Ala-Gly-(pNO2)Phe-Gly, leucine aminopeptidase, and phosphoramidon were obtained from Sigma. Human big ET-1 was purchased from Peptides International (Louisville, KY). All other peptides were purchased from Bachem Bioscience, Inc. (Torrance, CA). Polyoxyethylene-10-lauryl ether (C12E10) was from Calbiochem (La Jolla, CA).

Identification of Peptide Hydrolysis Products-- Peptides (0.25 mM) were incubated with solECE-1 (83 nM) at 37 °C in 50 mM MES-KOH, 0.01% C12E10, pH 6.5, for 2 to 16 h. Reaction products were separated by reversed-phase high performance liquid chromatography (HPLC) using pumps, ultraviolet detector, and software from Rainin Instrument Co. (Emeryville, CA). Peptides were bound to a 0.46 × 25-cm C18 column (Vydac, Hesperia, CA) and eluted by a gradient of 0 to 60% (v/v) acetonitrile in 0.1% trifluoroacetic acid. Peptides were detected by absorbance at 215 nm. Products were collected and analyzed by matrix-assisted laser desorption/ionization mass spectrometry. When necessary, products were further subjected to liquid chromatography-mass spectrometry or NH2-terminal amino acid sequence analysis to confirm their identity. SolECE-1 hydrolysis of dansyl-D-Ala-Gly-(pNO2)Phe-Gly and glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide were assayed fluorimetrically at concentrations of 0.1 mM as described previously for the assay of neprilysin (17).

Mass Spectrometry-- Matrix-assisted laser desorption/ionization mass spectra were acquired on a PerSeptive Biosystems, Inc. (Framingham, MA) Voyager Elite-Delayed-Extraction time-of-flight mass spectrometer. Radiation from a Laser Science, Inc. (Newton, MA) nitrogen laser (337 nm, 3-ns pulse width) was used to desorb ions from the target. All linear delayed extraction experiments were performed using an extraction grid voltage of 23.125 kV and a pulse delay of 150 ns. Twenty-five to 100 laser shots were averaged for each spectrum. Electrospray ionization mass spectra were acquired with a Finnigan MAT 900 double-focusing mass spectrometer. Analyses were performed at 5 kV full accelerating potential. Tandem mass spectra were acquired by scanning the magnet and the electrical analyzer simultaneously at a constant B/E ratio.

Determination of Kinetic Constants-- The rates of substrate hydrolysis were determined by measuring the appearance of products by HPLC under initial rate conditions (less than 10% hydrolysis of substrate, except for big ET-1). To detect products of big ET-1 hydrolysis at low substrate concentrations, it was necessary to digest up to 20% of the substrate. Depending on the substrate, solECE-1 concentrations ranging from 4.2 to 250 nM were used. Reactions were carried out in 50 mM MES-KOH, 0.01% C12E10, pH 6.5, at 37 °C. Reaction volumes ranging from 0.10 to 1.0 ml were analyzed by HPLC, depending on the detection limits of the products of interest. Product peak areas determined using Dynamax software (Rainin) were converted to mol of product using standard curves generated from known amounts of the peptide products. Big ET-1 hydrolysis was quantitated by measuring the formation of the COOH-terminal fragment, big ET-1 (22-38). The rates of hydrolysis of angiotensin I, angiotensin I (1-6), and bradykinin were measured by quantitating the appearance of the products angiotensin I (1-7), angiotensin I (1-4), and bradykinin (1-7), respectively. The rates of neurotensin and substance P cleavage were determined by measuring the simultaneous appearance of neurotensin fragments (1-10) and (3-13) and substance P fragments (1-6) and (1-9), respectively. The rate of hydrolysis of rat atrial natriuretic peptide (ANP) was quantitated by calculating the disappearance of the substrate. The concentrations of all substrates and peptide standards used were determined by quantitative amino acid analysis.

Initial velocity data (Vo) were plotted as a function of substrate concentration and fit to the Michaelis-Menten equation: Vo = Vmax [S]/(Km + [S]) using KaleidaGraph software (Synergy Software, Reading, PA) to obtain Km and Vmax values. Turnover numbers (kcat) were calculated using the equation kcat = Vmax/[E], using a subunit molecular mass of 1.2 × 105 Da for solECE-1. For the substrates angiotensin I (1-6) and rat ANP, Km was too high to be accurately measured, so the ratio kcat/Km was estimated from initial velocity data at low substrate concentrations using the equation (kcat/Km) Vo/([E][S]), a simplification of the Michaelis-Menten equation that applies when [S] << Km (18).

    RESULTS

The recombinant solECE-1 used in this study was purified to homogeneity from Chinese hamster ovary K1 cell-conditioned medium. The recombinant secreted enzyme was previously found to be indistinguishable from the native membrane-bound ECE-1 by a number of criteria (32). SolECE-1 hydrolyzed big ET-1 exclusively at the Trp21-Val22 bond with a specific activity comparable with the native enzyme. SolECE-1 was also inhibited by phosphoramidon and PD 069185 with Ki values comparable with those determined for the native enzyme. PD 069185 is a trisubstituted quinazoline compound that is a specific competitive inhibitor of ECE-1 (19).

Small Synthetic NEP Substrates Are Poor ECE-1 Substrates-- In an initial comparison of the substrate specificities of ECE-1 and NEP, two synthetic peptides commonly used for the assay of NEP were tested for hydrolysis by solECE-1. When solECE-1 was used at concentrations up to 83 nM for 16 h at 37 °C in the presence of 0.1 mM dansyl-D-Ala-Gly-(pNO2)Phe-Gly, no product was detected. Under the same conditions, except for the use of leucine aminopeptidase in a coupled assay, hydrolysis of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide was barely detectable, with a specific activity of less than 1.0 pmol/h/mg. In control experiments, recombinant NEP (kindly provided by Dr. P. Crine, University of Montreal) efficiently hydrolyzed both substrates.

solECE-1 Hydrolyzes a Variety of Bioactive Peptides-- An initial screen of potential ECE-1 substrates employed 12 biologically active peptides of varying sizes and structures using a relatively high enzyme concentration (83 nM) and a prolonged period of digestion (16 h). These peptides were chosen because of their biological activities (many are known to be vasoactive) and/or they have been characterized as substrates of NEP. Substrate hydrolysis was monitored by HPLC with the results summarized in Table I. No hydrolysis of neuropeptide Y or Arg8-vasopressin was detected. Leucine-enkephalin was an extremely poor substrate, as less than 5% of the peptide was hydrolyzed in the assay. Luteinizing-hormone-releasing-hormone and vasoactive intestinal peptide were also found to be poor substrates, with less than 25% hydrolysis observed under these conditions. The relatively large peptides calcitonin (32 residues) and rat ANP (28 residues) were both hydrolyzed to a significant degree. HPLC resolved at least 13 distinct product peaks for calcitonin and at least six for ANP, indicating cleavage at multiple sites by ECE-1. The smaller (9-13 amino acids) peptides angiotensin I, bradykinin, neurotensin, and substance P were all digested to completion or near completion. Oxidized insulin B chain was also hydrolyzed to completion, with 15 product peaks resolved by HPLC under these conditions. SolECE-1 hydrolysis of all peptides tested was completely inhibited by both 150 µM phosphoramidon and 150 µM PD 069185. Inhibition of solECE-1 hydrolysis of the oxidized insulin B chain by PD 069185 is shown in Fig. 1.

                              
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Table I
solECE-1 hydrolysis of biologically active peptides
Enzyme digests contained 0.25 mM peptide and 83 nM purified solECE-1 in 50 mM MES-KOH, 0.01% C12E10, pH 6.5. Control samples were identical, except that solECE-1 was omitted. Samples were incubated for 16 h at 37 °C before analysis by HPLC as described under "Experimental Procedures." Percent hydrolysis was determined by comparing substrate peak areas of control and solECE-1-digested samples.


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Fig. 1.   solECE-1 hydrolysis of the oxidized insulin B chain and inhibition by PD 069185. A, a 0.1-ml sample of oxidized insulin B chain (0.1 mM) in reaction buffer (50 mM MES-KOH, 0.01% C12E10, 1.5% Me2SO, pH 6.5) was analyzed by HPLC as described under "Experimental Procedures." B, oxidized insulin B chain (0.1 mM) was reacted with 4 nM solECE-1 in reaction buffer for 6 h at 37 °C. A 0.1-ml aliquot of this reaction was analyzed by HPLC, and the products were identified as described under "Experimental Procedures." Peak 1 was identified as a mixture of Ala-Leu (residues 14-15) and Pro-Lys-Ala (residues 28-30). Peak 2 is the NH2-terminal fragment (1-10) of the oxidized insulin B chain. Peaks 3-6 were identified as the insulin B chain fragments (17-27), (1-11), (1-14), and (17-30), respectively. Peak 7 is a mixture of insulin B chain fragments (1-16) and (16-30). Products eluting between 10-45% acetonitrile are shown. C, HPLC analysis of a 0.1-ml aliquot of a reaction identical to the one shown in B but containing 150 µM PD 069185, a specific inhibitor of ECE-1. The peak designated X is an unidentified component of the PD 069185 preparation. The two peaks designated as Y are unidentified impurities present in the oxidized insulin B chain preparation.

Determination of Cleavage Sites of Biologically Active Peptides by ECE-1-- The peptides identified as the best substrates in the initial screen were selected for further analysis to identify the sites of cleavage by ECE-1. These peptides were digested with solECE-1 for varying periods of time, and the products at each time point were analyzed by HPLC to identify the initial products and those that appear only after prolonged digestion. Product peaks were collected, and the peptides were identified by mass spectrometry and by NH2-terminal amino acid sequence analysis when necessary. Oxidized insulin B chain is not biologically active but was chosen for analysis because it is useful for mapping the peptide bond specificity of peptidases. The seven product peaks resolved by HPLC (see Fig. 1) were analyzed by mass spectrometry to determine the principal sites of cleavage of this peptide. The two small peaks designated as Y were not analyzed further because they appeared to be contaminants of the insulin B chain preparation. From time course studies it was evident that the peptide was initially cleaved at the Tyr16-Leu17 bond. The resulting Leu17-Ala30 fragment was relatively resistant to further hydrolysis, although cleavage at Thr27-Pro28 was detected. The Phe1-Tyr16 fragment was rapidly degraded to smaller peptides, principally by cleavage at the amino side of hydrophobic residues clustered between Leu11 and Leu15. Hydrolysis of the Leu15-Tyr16 bond was also detected early in the digestion of the oxidized insulin B chain. These data are summarized in Fig. 2.


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Fig. 2.   Principal sites of solECE-1 hydrolysis of oxidized insulin B chain. The peptide was digested with solECE-1, and the seven products identified in Fig. 1 were purified by HPLC and identified by mass spectrometry as described under "Experimental Procedures." The peptide sequence is designated by the one-letter code for amino acids. Cysteic acid residues are denoted by C*. The numbers above the peptide sequence represent the molecular masses (M + H+, in Da) of products of the digest as determined by mass spectrometry. The brackets spanning portions of the amino acid sequence denote the sequence assignment of digestion products based on molecular mass. The arrows mark the sites of cleavage by solECE-1 inferred from the product masses. Arrows marked by asterisks are sites of NEP hydrolysis of the oxidized insulin B chain previously determined (25).

ANP is a 28-amino acid peptide containing a ring structure formed by a disulfide bridge between Cys7 and Cys23. ANP was initially attacked by solECE-1 within the ring structure at the Cys7-Phe8 bond and at the Ser25-Phe26 bond (data not shown). Both of these bonds have been shown to be rapidly cleaved by NEP (31). Prolonged digestion of ANP by solECE-1 revealed that the Ser6-Cys7, Arg11-Ile12, and Cys23-Asn24 bonds were also hydrolyzed.

Less complicated patterns of cleavage by solECE-1 were observed for the shorter peptides examined. Bradykinin was hydrolyzed exclusively at the Pro7-Phe8 bond, as previously shown by Hoang and Turner (20). Angiotensin I was cleaved primarily at the Pro7-Phe8 bond, but hydrolysis at about 5% of this rate was observed at both Val3-Tyr4 and Tyr4-Ile5. Cleavage of angiotensin I at the Phe8-His9 bond was not observed, indicating that ECE-1 is not able to produce the vasoconstrictor peptide angiotensin II. The derivative angiotensin I (1-6) was hydrolyzed exclusively at the Tyr4-Ile5 bond to release the COOH-terminal dipeptide Ile-His. Neurotensin was hydrolyzed at both Pro10-Tyr11 and Leu2-Tyr3, with the Pro10-Tyr11 cleavage occurring at about 10 times the rate of Leu2-Tyr3 cleavage at all substrate concentrations tested. Once formed, the neurotensin (1-10) and (3-13) products were resistant to further hydrolysis by solECE-1. Substance P was rapidly cleaved at both Gln6-Phe7 and Gly9-Leu10; hydrolysis of the Gln6-Phe7 bond occurred at twice the rate of Gly9-Leu10 hydrolysis. The product substance P (1-9) was further hydrolyzed to generate substance P (1-6) and the tripeptide Phe-Phe-Gly through hydrolysis of the Gln6-Phe7 bond. After prolonged periods of digestion, hydrolysis of substance P at the Phe7-Phe8 bond was also observed. These results are diagrammed in Fig. 3 along with a comparison to the sites of cleavage of these peptides by NEP.


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Fig. 3.   Hydrolysis of biologically active peptides by solECE-1. The peptides were digested with solECE-1, and the resulting products were identified as described under "Experimental Procedures." The names of peptides analyzed are shown to the left of their amino acid sequences in one-letter code. pE represents a pyroglutamic acid residue. The numbers above the peptide sequences represent the molecular masses (M + H+, in Da) of products of the digest as determined by mass spectrometry. The brackets spanning portions of the amino acid sequences denote the sequence assignment of digestion products based on molecular mass. The arrows mark the sites of cleavage by solECE-1 inferred from the product masses. Principal sites of hydrolysis are shown by the larger arrows. Minor sites (representing less than 5% total hydrolysis by ECE-1) are shown by the smaller arrows. The arrows marked by asterisks represent sites in these peptides that are also hydrolyzed by NEP. The data for hydrolysis of these peptides by NEP were taken from Gafford et al. (27) and Skidgel et al. (29).

Kinetic Analyses-- solECE-1 hydrolysis of the peptides angiotensin I, bradykinin, neurotensin, and substance P was further characterized by the determination of the kinetic constants Km and kcat. Initial rate data (Vo) were obtained by HPLC analysis, plotted as a function of substrate concentration and fit to the Michaelis-Menten equation. As shown in Fig. 4, these four peptides exhibit saturation kinetics, and the initial rate data fit well to the Michaelis-Menten equation. The kinetic constants calculated from these data are summarized in Table II, where they are compared with values obtained for big ET-1, an in vivo substrate of ECE-1. Km values were found to vary greatly among the substrates examined, with the lowest (2.0 µM) found for big ET-1. Substance P and neurotensin were found to have moderately low Km values of 90 and 78 µM, respectively. Relatively high Km values of 0.34 and 2.5 mM were obtained for bradykinin and angiotensin I. Angiotensin I (1-6) was the smallest peptide hydrolyzed by solECE-1 at a significant rate. The Km for the hexapeptide angiotensin I (1-6) was too high to measure because initial velocity was linear with respect to substrate concentration through 10 mM. The Km of ANP could not be accurately determined because initial velocity was proportional to substrate concentration through 0.5 mM. The kcat values did not vary as much as those for Km. For solECE-1 hydrolysis of neurotensin and substance P, kcat values of 0.66 and 0.73 s-1, respectively, were determined. For angiotensin I, a kcat of 4.9 s-1 was obtained. The highest value of kcat, 23 s-1, was observed for solECE-1 hydrolysis of bradykinin. The high kcat of bradykinin gave this peptide the highest kcat/Km ratio of any substrate examined, including big ET-1 (kcat = 0.052 s-1).


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Fig. 4.   Initial velocity dependence on substrate concentration for solECE-1 hydrolysis of bioactive peptides. Initial velocity (Vo) data were determined for substance P, neurotensin, bradykinin, and angiotensin I as described under "Experimental Procedures." Substrate concentrations are plotted on the x axes; initial rates are plotted on the y axes as nmol of substrate hydrolyzed h-1. The data were fit to the Michaelis-Menten equation. Substrate concentrations were varied over the range 0.2-5.0 × Km for each peptide, except for angiotensin I, which was limited by substrate solubility. The concentration of solECE-1 was kept constant within each series of reactions.

                              
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Table II
Kinetic constants for solECE-1 hydrolysis of biologically active peptides
Measurements were made as described under "Experimental Procedures." Km and kcat values are reported ± S.E. ND, not determined.


    DISCUSSION

ECE-1 was originally purified as a phosphoramidon-sensitive metallopeptidase specific for the conversion of big ET-1 to the active vasoconstrictor ET-1. Despite the apparent structural similarity between NEP and ECE-1, a number of differences have been observed in their substrate specificity and sensitivity to inhibitors. For example, ECE-1 is insensitive to inhibition by thiorphan, a potent inhibitor of NEP (3). Endothelins are rapidly degraded by NEP (21) but are not hydrolyzed further by ECE-1 after their processing from big ET precursors. NEP has a well documented COOH-terminal dipeptidase activity, yet ECE-1 has been reported to require COOH-terminal-extended substrates (up to P10') for activity2 (8, 23). These and other studies led to the idea that ECE is a highly selective peptidase, in contrast to NEP, which has a broad substrate specificity (3). In this study we have examined the activity of purified recombinant solECE-1 against a number of peptide substrates and conclude that in contrast to previous assertions, ECE-1 exhibits a substrate specificity similar to that of NEP, preferentially hydrolyzing peptide bonds with a hydrophobic P1' residue. However, significant differences in activity against small peptides and in kinetics of hydrolysis of common substrates were observed between the two enzymes.

Initial experiments using purified solECE-1 showed that the enzyme had little or no detectable activity against two small synthetic peptides (glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide and dansyl-D-Ala-Gly-(pNO2)Phe-Gly) that are commonly used for the assay of NEP. Hydrolysis by solECE-1 of the pentapeptide Leu-enkephalin, another excellent NEP substrate, was barely detectable. However, significant solECE-1 activity was observed using the hexapeptide angiotensin I (1-6). Thus it appeared that substrates smaller than hexapeptides were not efficiently hydrolyzed by ECE-1. This was a significant difference from NEP, which is known to hydrolyze small peptides such as enkephalins and the chemotactic peptide formyl-Met-Leu-Phe in vivo (24).

When larger peptides were used as substrates, similarities in the substrate specificities of ECE-1 and NEP became readily apparent. Of the seven major sites of ECE-1 cleavage of the oxidized insulin B chain, five were found to coincide with sites previously reported for NEP (25). All five of these bonds were located on the amino side of hydrophobic residues, indicating that both enzymes preferentially hydrolyze this type of bond. This pattern was observed repeatedly in the analysis of ECE-1 hydrolysis of biologically active peptides, as the major sites of cleavage occurred predominantly at sites with Phe, Tyr, and Leu in the P1' position. In most cases these sites correspond to the major sites of NEP hydrolysis of these peptides. ECE-1 hydrolysis at sites with a Pro residue at P1 was observed with angiotensin I, bradykinin, and neurotensin as substrates, but it is not clear from this study whether Pro is preferred at this position because sites containing Gly, Gln, and Leu residues at P1 were also efficiently cleaved. In this respect ECE-1 is similar to NEP, which also tolerates a variety of residues in the P1 position (26). It is notable that for three peptides, angiotensin I (1-6), bradykinin, and substance P, solECE-1 was found to remove a COOH-terminal dipeptide. This dipeptidase activity was unexpected in light of previous reports that ECE-1 requires an extended COOH-terminal sequence for hydrolysis of big ET-1 analogs. It is possible that the previous observations are a result of features unique to big ET-1 and ET-1, where their COOH-terminal structures may prevent cleavage by ECE-1. It could be argued that the recombinant solECE-1 used in this study has undergone a structural change that alters its normally restricted substrate specificity. However, this seems unlikely because solECE-1, like the native enzyme, cleaved big ET-1 exclusively at the Trp21-Val22 bond and did not hydrolyze the resulting products. Furthermore, the dipeptidase activity against bradykinin was previously reported using the native membrane-bound form of ECE-1 also expressed in Chinese hamster ovary K1 cells (20).

Significant differences between NEP and ECE-1 were noted in the kinetics of hydrolysis of the substrates used in this study. The Km values determined in this study for ECE-1 hydrolysis of bradykinin, neurotensin, and substance P did not vary more than 3-fold from the values previously determined for NEP hydrolysis of these peptides (27, 28). However, the higher rate of NEP hydrolysis of these three peptides is reflected in kcat/Km ratios that are 10-, 32-, and 320-fold greater for bradykinin, neurotensin, and substance P, respectively. The kcat value for angiotensin I is similar for both NEP and ECE-1, but the weak binding to ECE-1 (Km = 2.5 mM) makes it a poor substrate when compared with NEP (Km = 36 µM (29)). ANP is an excellent substrate for NEP (30) but is not hydrolyzed efficiently by ECE-1, as kcat/Km was estimated to be 250-fold lower. None of the peptides tested in this study were found to bind to ECE-1 as well as big ET-1 (Km = 2.0 µM), but the kcat/Km values of neurotensin and substance P were comparable with that of big ET-1, and the kcat/Km of bradykinin exceeded the big ET-1 value. A possible explanation of this is that extended COOH termini promote the binding of peptides to ECE-1 but that product release then becomes rate-limiting for larger substrates such as big ET-1. This effect is not evident in catalysis by NEP, which binds both large and small peptides with high affinity and hydrolyzes them with comparable kcat values.

It is noteworthy that ECE-1 hydrolyzes bradykinin at a rate greater than the in vivo substrate big ET-1. This cleavage of bradykinin at the Pro7-Phe8 bond would destroy its vasodilatory actions in endothelial tissue. Because a major site of ECE-1 expression is the endothelium, it is possible that ECE-1 could function in a manner analogous to angiotensin I-converting enzyme by both producing a potent vasoconstrictor and inactivating a potent vasodilator. A recent study described the effects of the targeted disruption of the ECE-1 gene in mice (10). ECE-1 null embryos exhibited developmental malformations highly similar to those caused by disruptions of genes encoding the endothelins and endothelin receptors. Only a deficiency in big ET-1 processing, but no other defects in peptide hormone processing, were reported. However, the ECE-1 null mice died in utero or shortly after birth, so it was not possible to observe the effects of ECE-1 deficiency at later stages of development. ECE-1 is expressed in many adult tissues, and its related isoform ECE-2 appears to be expressed in the secretory pathway, particularly in neural tissue (13). Therefore ECE would be exposed to many potential substrates in vivo. Given its broad substrate specificity, it is likely that ECE may be involved in the degradation and processing of many biologically active peptides, both at the cell surface and in the secretory pathway.

    ACKNOWLEDGEMENT

We thank Dr. Robert Fuller for his critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-5903; Fax: 734-622-1355; E-mail: Kay.Ahn{at}aa.wl.com.

The abbreviations used are: ET, endothelin; ANP, atrial natriuretic peptide; C12E10, polyoxyethylene-10-lauryl ether; dansyl, 5-(dimethylamino)naphthalene-1-sulfonyl; ECE, endothelin-converting enzyme; HPLC, high performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; NEP, neprilysin, neutral endopeptidase 24.11; (pNO2)Phe, para-nitro-L-phenylalanine; solECE-1, soluble ECE-1.

2 For a thorough explanation of this subsite notation for peptidases and their substrates, please see Schechter and Berger (22).

    REFERENCES
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Abstract
Introduction
References

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