From the Department of Biochemistry and
§ Department of Chemistry, Parke-Davis Pharmaceutical
Research, Division of Warner-Lambert Company,
Ann Arbor, Michigan 48105
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ABSTRACT |
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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.
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 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.
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] 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.
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.
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.
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 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.
INTRODUCTION
Top
Abstract
Introduction
References
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).
EXPERIMENTAL PROCEDURES
Km (18).
RESULTS
solECE-1 hydrolysis of biologically active peptides
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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.
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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).
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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).
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.
Kinetic constants for solECE-1 hydrolysis of biologically active
peptides
DISCUSSION
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ACKNOWLEDGEMENT |
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We thank Dr. Robert Fuller for his critical reading of the manuscript.
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FOOTNOTES |
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* 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).
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REFERENCES |
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