(Hypertension. 2000;36:116-a.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Effects of the N-Terminal Sequence of ACE on the Properties of Its C-Domain
From the Departments of Pharmacology (B.M., P.A.D., H.L.J., E.G.E., F.T.) and Anesthesiology (E.G.E., F.T.), University of Illinois College of Medicine at Chicago.
Correspondence to Ervin G. Erdös, MD, Dept of Pharmacology (M/C 868), University of Illinois-Chicago, 835 S Wolcott Ave, Chicago, IL 60612. E-mail EGErdos{at}uic.edu
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Abstract |
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Abstract—Angiotensin I–converting enzyme (ACE, kininase II) has 2 active domains (N and C) in a single peptide chain. Because we found its N-domain more stable than its C-domain, we investigated the effect of the amino-terminus of human ACE on the C-domain with a molecular construct expressed in Chinese hamster ovary cells (CHO) cells and transiently in HEK293 cells. This active N-deleted ACE contained only the first 141 amino acids of the human N-domain but not its active center and was linked to the active C-domain containing the transmembrane and cytosolic portions of ACE. The CHO cells were also transfected with human B2 bradykinin receptor. ACE inhibitors (5 nmol/L or 1 µmol/L) augmented bradykinin (100 nmol/L) effects, elevated B2 receptor numbers, and resensitized the receptor desensitized by agonist as measured by arachidonic acid release or [Ca2+]i mobilization. Arachidonic acid release was mediated by pertussis toxin–sensitive G
i, and
[Ca2+]i mobilization was mediated by
pertussis-insensitive Gq protein receptor
complex. The properties of the construct were compared with wild-type
ACE and separate N- and C-domains. The N-deleted ACE differed from
wild-type in activation by Cl- and
[SO4]2- ions, hydrolysis ratios of
substrates (both short synthetic and endogenous peptides)
and heat stability. Thus, the N-terminal peptide of ACE affected the
characteristics of the C-domain active center. ACE
inhibitors acting on N-deleted ACE, which had only a single
C-domain active center anchored to plasma membrane, induced cross-talk
between the enzyme and the B2 receptor (eg, the
inhibitors resensitized the receptor) independent of
blocking bradykinin inactivation.
Key Words: angiotensin • bradykinin • angiotensin-converting enzyme inhibitors • receptors, bradykinin • arachidonic acid • calcium
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Introduction |
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Angiotensin I–converting enzyme (ACE, or kininase II) is a single-chain protein with 2 domains (N- and C-domain) named according to their proximity to the N- or C-terminal end.1 2 The 2-domain, widely distributed enzyme is called somatic ACE, in contrast to the testicular ACE (t-ACE), which represents the C-domain with an additional N-terminal sequence.3 This originates from an alternate initiation site in intron 12 of the ACE gene.4 The synthesis of both enzymes is directed, however, by a single gene. Although the homogeneity around the active centers is
89%,4 the 2 active sites cleave some substrates at
different rates. In addition to active centers and substrate binding
sites, the positions of all 7 cysteine residues in each domain are
highly conserved (there are no cysteine residues in the other regions
of the molecule), which indicates that both domains have some
similarity in topological structure. The most important substrates,
bradykinin (BK) and angiotensin (Ang) I, are hydrolyzed at
both sites,2 5 but some of the others are cleaved
preferentially by 1 of the sites, which is frequently on the N-domain.
Of peptide hormones, for example, the luteinizing hormone-releasing
hormone (LHRH) is inactivated mostly by the release of
<Glu1-His2-Trp3,6 7
and the enkephalin precursor
Met5-Enk-Arg6-Phe7
is converted primarily to enkephalin by the N-domain
site.8 9 Ang 1-710 and the tetrapeptide
AcSer-Asp-Lys-Pro (AcSDKP)11 12 are also cleaved by the
N-domain. Although the role of individual amino acids in the active center has been well studied,4 the function of the N-terminal sequence in preserving the integrity of the N-domain of ACE is not known. We have been puzzled by our findings that although human intestinal brush border is quite rich in somatic ACE,13 ileal fluid contains the highly active intact N-domain of ACE only, but no C-domain is detected there.14 This separate N-domain has an estimated sequence from 1to 443 amino acids. Thus, we questioned whether the N-terminal end preceding the active center of the N-domain would lend stability to ACE in the protease-rich milieu of the intestine. The integrity of the C-domain attached to plasma membrane can be protected by the N-domain on 1 side and by the transmembrane anchor portion of ACE on the other side.1 15 To learn more on the importance of structural elements in ACE, we constructed an ACE molecule that contained the first 141 amino acids of the N-domain without an active center (N-del ACE) and coupled it to the C-domain of human ACE, having the active center and the transmembrane and cytosolic portions of the wild-type ACE (WT-ACE).4 8 Because the 2 domains of ACE differ in substrate specificity and presumably in stability, we investigated how these properties are affected by the N-terminal peptide chain, ie, whether, in addition to the individual amino acids in and around the active center, other factors may influence ACE activity. We also wanted to establish whether ACE inhibitors can still potentiate BK effects on its receptor by acting on N-del ACE, having only the C-domain active site.
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Methods |
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Materials BK, Ang, Ang 1-7, AcSDKP, hippuryl-His-Leu (Hip-His-Leu), tissue culture medium, buffers, reagents, and gel filtration columns were obtained from Sigma Chemical Co or from Pharmacia. Fetal bovine serum (FBS) was obtained from Atlanta Biologicals. Benzyloxycarbonyl (Z)-Phe-His-Leu was obtained from Bachem. Enalaprilat and lisinopril were provided by the Merck, Sharp and Dohme Research Division (WesPoint, Pa). Keto-ACE (5 S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline) was a gift from Dr R.G. Ahlquist at the Stanford Research Institute (Palo Alto, Calif). The wild-type cDNA of ACE was kindly donated by Prof P. Corvol, College de France, Paris, France. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells were from the American Type Culture Collection. Transfection reagents were from Qiagen. The human active, separate N-domain and C-domain and WT-ACE and rabbit t-ACE were prepared as described.3 10 14
Culture and Transfection of CHO Cells
Cells were grown and transfected as previously
described.10 16 17 CHO cells were plated at density
1x105 cells per 60-mm dish 1 day before
transfection.10 16
Culture and Transfection of HEK293 Cells
HEK293 cells were grown via the same general culture procedures
used for CHO cells. Transient transfection of HEK293 cells was
performed with SuperFect Transfection reagent.
Construction of N-del ACE
There are 2 Bgl II restriction enzyme cleavage sites (A/GATCT)
in the ACE cDNA sequence at 533 bp and 2327 bp. This 1.8 kb Bgl II
fragment codes for much of the N-domain, including the active site, the
bridge peptide14 that connects N- and C-domain of ACE, and
a small portion of C-terminal active domain. The protein sequences
around the 2 Bgl II sites are very similar: LDPDLTN versus LEPDLTN.
When the Bgl II fragment was removed, there was no shift of the reading
frame.
The 1-532 Eco RI- Bgl II and 2328-4020 Bgl II- Eco RI fragments of ACE cDNA were ligated into the dephosphorylated Eco RI site of pcDNA3 vector (Invitrogen). The ACE-Bgl II deletion-pcDNA3 transfer vector DNA was purified for the transfection of CHO or HEK293 cells.
This deleted form of the ACE molecule (N-del) contains the signal peptide, the N-terminal region, and the C-domain, including the stem peptide, the transmembrane anchor peptide and the cytoplasmic C-terminus. The N-domain contributes 2 cysteine residues, and the C-domain supplies 5 cysteine residues (Figure 1).
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Transfection With Human B2 Receptor cDNA
The selected clones were transfected with pcDNA3 vector
containing human B2 BK receptor cDNA by use of
the Superfect transfection method. The cells expressing both ACE and
B2 receptors were marked CHO/AB.
Radioligand Binding on Selected Clones
To select the clone with the highest expression of
B2 receptors, [3H]BK
saturation binding was performed on whole CHO/AB cell monolayers.
Clones with high expression of B2 receptors were
used further.16
Effect of Pertussis Toxin
CHO cells expressing WT-ACE and B2
receptor were incubated with 500 ng/mL pertussis toxin in Ham’s F-12
medium at 37°C for 18 hours.18 19 Afterwards, either
3H arachidonic acid (AA) release
induced by 100 nmol/L BK or 100 nmol/L BK+1 µmol/L
ramiprilat or the effect of BK and ramiprilat
on [Ca2+]i was
measured.
Enzyme Solubilization and Assay
Transfected HEK293 or CHO/AB cell monolayers expressing ACE were
washed twice with PBS and were then suspended by scraping into 50
mmol/L Tris maleate buffer (pH 7.4) plus 0.5% CHAPS detergent. The
cell suspension was incubated at 4°C for 18 hours and was then
centrifuged at 100 000g for 1 hour. The
supernatant, which contained the detergent solubilized enzyme, was then
assayed.
In general, enzymatic activity of ACE was assayed with Hip-His-Leu or Z-Phe-His-Leu. The released His-Leu was measured after adding 100 µL of 20 mg/mL o-phthaldialdehyde.17
Hydrolysis of Peptides
The rates of hydrolysis of BK and Ang were generally determined
at 37°C in 50 mmol/L Tris-maleate buffer (pH 7.4) containing
150 mmol/L NaCl, 100 µmol/L substrate, and 10 µL of
solubilized enzyme from HEK293 cells in the absence or presence of
1.0 µmol/L enalaprilat.
The hydrolysis of the various peptides by the ACE enzymes was assayed by high-pressure liquid chromatography. It was established that cleavage of the substrates followed zero-order kinetics to the time point of the assay.9 20
Effect of Cl– on Activity
The hydrolysis of Z-Phe-His-Leu by solubilized enzymes was
measured in NaCl added to assay buffer in concentrations ranging from 0
to 800 mmol/L.
Effect of [SO42-] on Activity
The hydrolysis of Hip-His-Leu was measured in assay buffer
containing 150 mmol/L NaCl and either 0 or 900 mmol/L
Na2SO4.
Keto ACE Inhibition
The inhibition of the enzymatic hydrolysis of 1 mmol/L
Hip-His-Leu by keto-ACE was determined by preincubating the enzymes for
30 minutes at 4°C with
10-8 to
10-4 mol/L keto-ACE before
the addition of substrate.10
Measurement of [Ca2+]i and
[3H] AA
Free cytosolic calcium
[Ca2+]i was measured by
means of a microspectrofluorometer (PTI Deltascan or Attofluor
Ratiovision) with fura-2/AM reagent.17 In some
experiments, [Ca2+]i was
recorded in a single cell or in 100 cells
simultaneously by means of an Attofluor Ratiovision
microspectrofluorometer.5 [3H] AA
release was determined as described.5 17
Desensitization and Resensitization of the B2
Receptor
After desensitization of the receptor by initial exposure
of cells to BK (0.1 to 1.0 µmol/L), the restoration of
sensitivity to the agonist (resensitization) was established by
measuring either [3H] AA release or
mobilization of
[Ca2+]i.16,17 For
example, monolayers of transfected CHO cells loaded with
[3H] AA were stimulated with BK for 30
minutes. Then, without removal of BK from medium or the addition of
more BK, cells were exposed to an ACE inhibitor (5 nmol/L
to 1 µmol/L) or, as control, to BK for an additional 5 minutes.
AA release by or [Ca2+]i
mobilization in the cells was registered.
ACE-Derived Enzymes
To obtain separate active N- and C-domains, purified human
kidney ACE20 was digested with endoprotease AspN (Sigma)
for 18 hours at 37°C.21 The fragments were separated and
purified by differential elution from a
lisinopril-Sepharose column14 and by gel
filtration on a Superdex 200 FPLC column.
Heat Stability
To compare the heat stability, separate N-domain ACE, C-domain
ACE, N-del ACE, and WT-ACE were used. Enzyme samples were diluted 1:10
into 50 mmol/L Tris-maleate (pH 7.4) containing 150 mmol/L
NaCl and were incubated for 25 minutes at temperatures ranging from
25°C to 60°C. The enzymatic activity remaining in the aliquots was
then measured with Z-Phe-His-Leu.
Protein Assays
Protein concentrations were assayed as
before.16
Statistics
Probability values were calculated by the unpaired t
test by means of Slidewrite software (Advanced Graphics
Software).
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Results |
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Hydrolysis of Hip-His-Leu and Z-Phe-His-Leu
The ratio of rates of hydrolysis of Z-Phe-His-Leu to that of Hip-His-Leu22 was determined for the N-del ACE from HEK293 cells, WT-ACE, N-domain ACE, and t-ACE (Table 1). The N-del ACE hydrolyzed Z-Phe-His-Leu
11 times faster than it did Hip-His-Leu. This ratio
was 3 times larger than with N-domain ACE and 12 times larger than
with t-ACE. Clearly, inserting the C-domain into the site of the active
portion of the deleted N-domain created an enzyme that hydrolyzes
substrates at a different ratio than does WT-ACE, N-ACE, or t-ACE.
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Activation of B2 Receptor
The recombinant ACE with partial N-domain deletion (N-del ACE) has
the first 141 amino acids of the N-terminal end of ACE but is missing
most of the N-domain sequence (residues 142 to 739). The missing part
includes the active center and the so-called "bridge
section"14 connecting both domains. The CHO clone used
in the experiments expressed 60 000 B2 receptors
and 10 000 ACE molecules per cell.
The cells expressing both N-del ACE and B2 receptor were then tested with enalaprilat to potentiate the BK-induced [3H] AA release. Enalaprilat (5 nmol/L) potentiated BK-induced [3H]AA released 1.7±0.4-fold (n=3, P<0.05), whereas 1 µmol/L enalaprilat enhanced it 2.8±0.6-fold (n=3, P<0.05). Enalaprilat also resensitized the receptor when added to the cells desensitized by the agonist; it reactivated the receptor to the desensitizing dose of BK still present in the medium. Enalaprilat (5 nmol/L) increased the release of AA 3.4±0.9-fold (P<0.05), and 1 µmol/L enalaprilat increased it (7.4±1-fold) (n=3, P<0.005) compared with the effects of control buffer or a second inactive dose of BK taken as 1 (n=3; P<0.005).
The effect of enalaprilat on the B2 receptor number in CHO/AB cells was also tested. Pretreatment with 5 nmol/L enalaprilat increased the specific [3H]BK binding insignificantly, only an average of 1.2-fold (range 1.18 to 1.24), and 1 µmol/L enalaprilat elevated the [3H]BK binding sites 2.5-fold (range 2.34 to 2.68) (n=2). Because these results with CHO/AB cells were in agreement with those obtained with WT-ACE and B2, they were not pursued further.16
Another interaction between N-del-ACE and B2 receptors was also investigated by using ramiprilat to resensitize receptor to BK and measuring [Ca2+]i mobilization. Ramiprilat (1 µmol/L), the active form of another ACE inhibitor, resensitized the receptor that was desensitized to BK 1.37±0.21-fold (n=5) compared with the first response to BK. When instead of the ACE inhibitor a second dose of BK was given, it was inactive, as noted before.16 17 Obviously, this resensitization of the receptor to BK by ACE inhibitor cannot be explained by the inhibition of its enzymatic breakdown.
Consequently, the single active C-domain of N-del ACE anchored to plasma membrane was sufficient to mediate an interaction of ACE with B2 receptors induced by ACE inhibitor.
Effect of Pertussis Toxin
Treatment of CHO cells expressing both WT-ACE and
B2 with pertussis toxin to block protein
abolished the release of AA. Neither 100 nmol/L BK alone
(1.05±0.22-fold over basal rate) nor in combination with
ramiprilat (1 µmol/L) added to treated CHO cells
expressing WT-ACE and B2 receptor (Figure 2A) released any significant amount of AA
over baseline (n=3). Pertussis toxin, however, did not affect the
elevation of [Ca2+]i by
BK, which was stimulated 2.47±0.6-fold over basal level (n=3,
P<0.05). Furthermore, after the initial
B2 receptor response, the receptor desensitized
by BK was resensitized to the BK in the medium by 1 µmol/L
ramiprilat and increased
[Ca2+]i 3.4±0.4-fold
over basal level (n=3, P<0.05) (Figure 2B).
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Thus the BK-induced AA release and potentiation of this response by
ramiprilat are mediated by protein inactivated
by pertussis toxin. On the other hand, the BK-induced increase in
[Ca2+]i as well as the
resensitization of this response by ramiprilat are mediated
by the toxin-insensitive
Gq.19 23
Inhibition by Keto-ACE
Keto-ACE is a relatively specific inhibitor of the
C-domain active site of ACE.10 Therefore, the
effectiveness of this inhibitor was determined for the
N-del ACE as compared with N-domain and t-ACE. Keto-ACE inhibited the
N-del ACE similarly to t-ACE. The IC50 of
keto-ACE for N-del ACE (0.09±0.002 µmol/L) or t-ACE
(0.12±0.03 µmol/L) was much lower than the
IC50 for N-domain ACE (15±7.0 µmol/L;
P<0.005); thus, the inhibition of N-del ACE active center
did not differ from that in C-domain.
Activation of ACE Enzymes by Chloride (Cl–)
Chloride ions activate ACE24 25 but affect
the 2 active sites of ACE unequally.4 8 We tested the
effect of Cl– on N-del ACE activity between 0
and 800 mmol/L NaCl concentrations (Figure 3). Cl–
activates N-del ACE differently from either the isolated N- or
C-domain or WT-ACE. The N-del ACE has an optimum
Cl– of 200 mmol/L, and higher
Cl– concentrations inhibit hydrolytic activity.
WT-ACE reached a Cl– optimum at 800 mmol/L
(Figure 3).
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, WT-ACE.
Activation by Sulfate [SO42-]
Na2SO4 accelerates the
hydrolysis of Hip-Gly-Gly while inhibiting the cleavage of other
substrates by ACE.2 The activity of N-del ACE, t-ACE, and
N-domain ACE was measured with and without added 900 mmol/L
Na2SO4 with 1.0 mmol/L
Hip-His-Leu (Table 2).
Na2SO4 enhanced the
hydrolysis of Hip-His-Leu by the N-del ACE 3.3-fold and strongly
inhibited (80% or more) the cleavage by the other ACE congeners
tested, including WT-ACE, N-domain of ACE, and t-ACE.
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Hydrolysis of Natural Substrates
We compared the rates of hydrolysis of 4 naturally occurring
peptide substrates of ACE (Ang I, Ang1-7, BK, and Ac-SDKP) by both
N-del ACE (HEK293 cells) and WT-ACE (Table 3). N-del ACE cleaved AcSDKP poorly
(0.7±0.5 nmol/min per mg protein) relative to the other peptides
(30.8±4.7 nmol/min per mg for BK and 19.2±2.3 nmol/min per mg for Ang
I). However, N-del ACE cleaved BK faster than Ang I, and WT-ACE acted
in the opposite way (Ang I 38.1±7.0 nmol/min per mg and BK 21.0±4.9
nmol/min per mg).
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Ang 1-7 is cleaved by the N-domain of ACE to Ang 1-5 and a
dipeptide,10 but it inhibits the C-domain of ACE. In this
study, the C-domain in N-del ACE was active and cleaved Ang 1-7
(4.1±0.7 nmol ·
min-1 ·
mg-1 protein) 8 times
slower than BK (Table 3). Therefore, N-del ACE, in contrast to
the results obtained with C-domain ACE,10 acted similarly
to N-domain ACE and hydrolyzed Ang 1-7 at a significant rate, but not
Ac-SDKP, another substrate of the N-domain.
Heat Stability
The activity of the N- and C-domain of ACE and that of somatic
WT-ACE and N-del ACE was measured after they had been incubated 15
minutes at temperatures ranging from 25°C to 60°C (Figure 4). The stability of the various ACE
molecules differed considerably. C-domain ACE was the least stable; it
retained 23.3±2% activity after 15 minutes at 47°C. The N-domain
ACE was most stable (55±6% at 47°C), and WT-ACE, obviously
stabilized by N-domain, was similar to N-domain ACE (54±6% at
47°C). The N-del ACE was more sensitive to denaturation by heat than
either the N-domain or WT-ACE (43±4%) but was less sensitive than
C-domain alone (P>0.005).
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,
N-domain ACE; 
, C-domain ACE; 
, somatic ACE; •, N-del ACE.
Values are mean±SEM ( n=3). |
Discussion |
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We investigated how the N-terminal sequence of ACE, normally distal to cell membrane and facing body conduits, can change some properties of ACE and thus how structural factors can alter the functions of this single-chain, 2-domain enzyme. We constructed an ACE molecule to incorporate the first 141 amino acids of ACE, the mutant (N-del ACE) linked P141 of the N-domain to D740 in the C-domain, thus retaining the N-terminal end of ACE but only a single active center in the intact C domain (Figure 1). N-del ACE differs from WT-ACE, t-ACE, and the other congeners in substrate hydrolysis, in reactivity to anions, and in heat stability. For example, BK was inactivated relatively faster than Ang I by the N-del ACE, but with WT-ACE, the ratio was reversed (Table 3). Of the other endogenous substrates tested, AcSDKP was hydrolyzed very slowly by N-del ACE, because this tetrapeptide is a substrate primarily of the N-domain active center.11 12 Ang 1-7, a substrate of the N-domain but an inhibitor of the C domain of WT-ACE,10 was cleaved at an appreciable rate by this modified C-domain.
Z-Phe-His-Leu and Hip-His-Leu are hydrolyzed by both sites of ACE but
at different ratios of rates: 1.3 for the WT-ACE and 0.9 for
t-ACE.22 However, the ratio for N-del ACE was entirely
different; it cleaved Z-Phe-His-Leu 11 times faster than the t-ACE
representing the unmodified C-domain (Table 1).
ACE is a chloride-activated enzyme, but activation depends on the structure of the substrate.7 8 The hydrolysis of BK is enhanced less by Cl– than that of Ang I,24 25 although BK is cleaved more by the C-domain than by the N-domain. In somatic ACE, the N-domain active center is activated at much lower Cl– concentration (10 mmol/L) than that of the C-domain (up to 800 mmol/L).26 N-del ACE differed, because it was most active in 200 mmol/L NaCl, and higher concentrations of NaCl inhibited N-del ACE. In contrast, the optimal Cl– concentration for WT-ACE was 800 mmol/L.26
Added Na2SO4 also
distinguished N-del-ACE. Sulfate ions enhance the cleavage only of
substrates containing C-terminal Gly-Gly27 28 ; others are
inhibited by this anion. N-del-ACE, however, cleaved Hip-His-Leu 3
times faster in the presence of [SO42-],
and other ACE congeners were inhibited by 
80% by
Na2SO4 (Table 3).
When the inactivation of ACE by heating was determined, the isolated N-domain ACE was most stable, and the isolated C-domain was the most labile. N-del ACE was considerably more stable at 47°C than was the isolated C-domain.
The carbohydrate content of N-del ACE would also contribute some of the observed differences because the 1-141 sequence of N-del ACE contains 6 putative sites for N-linked carbohydrate in addition to the sites on the C-domain of ACE.4
ACE inhibitors (eg, ramiprilat, enalaprilat) potentiate the effects of BK and even of its ACE-resistant analogues on the B2 receptor indirectly.6 16 17 29 They are inactive on cells that have B2 receptor but lack ACE. This cross talk between the enzyme and the receptor is induced also by agents that react only with the N-domain17 active center. As shown, ACE inhibitors potentiated BK effects on B2 receptor in the cells that expressed N-del ACE. Consequently, inhibitors that react with either active center of ACE can augment the action of BK and its ACE-resistant analogues on the B2 receptor.16 17 This indirect activation of the B2 receptor by ACE inhibitors depends on the steric relationship of the enzyme to the receptor.29
The release of prostaglandins and nitric oxide are among
the most important indirect effects of BK30 31 ; they are
initiated by the complexing of the B2 receptor
with the Gi or
Gq protein.31 32 ACE
inhibitors augmented AA release at a lower
concentration16 17 than enhanced
[Ca2+]i elevation by BK.
The involvement of the 2 different G proteins in the activities of
B2 receptors was tested with pertussis
toxin19 23 in CHO WT-ACEB cells. The toxin abolished
the release of AA by the cells, by BK or by BK, and by ACE
inhibitor; thus, the activation of phospholipase
A2 was blocked. On the other hand,
[Ca2+]i
mobilization17 33 and its enhancement by ACE
inhibitor were not affected (Figure 2). The release
of [Ca2+]i, a consequence
of phospholipase C activation, depends on the complexing of the
receptor with Gq
protein.17 19
Our findings are consistent with the observations of Williams et al,22 who created chimeric N- and C-ACE domains in which 13 amino acids within the 60 central amino acid sequence that differ between the domains were exchanged for a corresponding sequence of the other domain. The hydrolysis rates of 2 substrates by the 2 domains indicated that this depended on the sequence surrounding the central regions in each domain rather than on the central regions themselves.
In conclusion, the properties of the C-domain active center were significantly altered by the added N-terminal peptide chain in N-del ACE. Thus, in addition to the amino acids in and around the active center, the structural elements of the N-domain can effectively influence activity at the C-domain.
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Acknowledgments |
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These studies were supported in part by NIH National Heart, Lung, and Blood Institute grants R01 HL-36473 and R01 HL-58118. The authors are grateful to Sara Bahnmaier for editorial assistance and Huan-Zhu Wang for technical assistance.
Received September 22, 1999; first decision November 24, 1999; accepted January 27, 2000.
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