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International Journal of Biological Macromolecules 74 (2015) 304–309

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

haracterization of angiotensin I-converting enzyme from anteriorills of the mangrove crab Ucides cordatus

atrícia A. Bersanetti a,∗, Regina F. Nogueiraa, Marcelo F. Marcondesb, Paulo B. Paivaa,aria A. Julianob, Luiz Julianob, Adriana K. Carmonab, Flavia P. Zanottoc

Departamento de Informática em Saúde, Universidade Federal de São Paulo, Rua Botucatu 862, São Paulo, SP, BrazilDepartamento de Biofísica, Universidade Federal de São Paulo, Rua Pedro de Toledo 669, São Paulo, SP, BrazilDepartamento de Fisiologia, Universidade de São Paulo, Rua do Matão, Travessa 14, n 321, São Paulo, SP, Brazil

r t i c l e i n f o

rticle history:eceived 18 November 2014eceived in revised form9 December 2014ccepted 20 December 2014vailable online 25 December 2014

eywords:rab gillCE isoform

a b s t r a c t

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates,invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE,here named crab-ACE, which presented catalytic properties closely resembling to those of mammalianACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogene-ity and a band of about 72 kDa could be visualized after silver staining and Western blotting. Assaysperformed with fluorescence resonance energy transfer (FRET) selective ACE substrates Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH, allowed us to verify that crab-ACE has hydrolytic profilevery similar to that of the ACE C-domain. In addition, we observed that crab-ACE can hydrolyze theACE substrates, angiotensin I and bradykinin. The enzyme was strongly inhibited by the specific ACE

haracterizationRET peptides

inhibitor lisinopril (Ki of 1.26 nM). However, in contrast to other ACE isoforms, crab-ACE presented a veryparticular optimum pH, being the substrate Abz-FRK(Dnp)-P-OH hydrolyzed efficiently at pH 9.5. Otherinteresting characteristic of crab-ACE was that the maximum hydrolytic activity was reached at around45 ◦C. The description of an ACE isoform in Ucides cordatus is challenging and may contribute to a betterunderstanding of the biochemical function of this enzyme in invertebrates.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Angiotensin I-converting enzyme (ACE) is a zinc-dependentetallopeptidase that hydrolyzes angiotensin I, releasing the

otent vasopressor angiotensin II [1] and inactivates the vasode-ressor bradykinin [2,3]. In mammals, ACE is associated with renins part of the homeostatic mechanism that controls blood pressure4,5] and electrolyte balance [6]. ACE is found as a somatic iso-orm (150–180 kDa) that contains two active sites within a singleolypeptide sequence [7,8] and a smaller isoform (90–110 kDa) that

s present exclusively in male germinal cells, apparently performingeproductive functions [9].

Components of the renin–angiotensin system (RAS) and ana-

ogues of ACE have been identified in many invertebrate species,laying roles in reproduction, osmoregulation, memory processesnd immune system regulation [10]. Isoforms of ACE can be found

∗ Corresponding author at: Departamento de Informática em Saúde, Universidadeederal de São Paulo, Rua Botucatu, 862, Térreo, São Paulo, SP, Brazil.

E-mail address: bersanetti@unifesp.br (P.A. Bersanetti).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.12.036141-8130/© 2014 Elsevier B.V. All rights reserved.

in annelids [11,12], crustaceans [13–17], molluscs [18], insects[19] and prokaryotes [20,21]. However, little is known aboutthe angiotensin system components in invertebrates, particularlyabout the role that ACE plays in this group.

In crustaceans, the enzyme presents high polymorphism and isfound as single or two domains forms in a variety of tissues [15].In these invertebrates, ACE seems to regulate the balance of bodyfluids during water deprivation [14] and may be responsible forthe degradation of crustacean hyperglycemic hormone involved incarbohydrate and osmotic balance [13].

Ucides cordatus is a strong osmotic regulator crab found inmangrove areas, which shows fluctuation in water salinity thatvaries from freshwater to almost full seawater. To survive in theseenvironments, this crab presents morphological and physiologicaldifferences in their gills [22]. The anterior gills have a thin epithe-lium to allow gas exchange, performing a respiratory function. Onthe other hand, the posterior gills have a thick epithelium that

facilitates ion exchange, presenting ion transporters at the plasmamembrane [23]. As very little is known about ACE in crustaceans,the objective of this work was to describe and characterize an ACEisoform from anterior gills of the crab Ucides cordatus, aiming to

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urther understand the biochemical properties of this enzyme innvertebrates.

. Material and Methods

.1. Animals

The crabs (Ucides cordatus) were collected at Itanhaém in Sãoaulo Coast (Lat. 24◦10′59′′S; Long. 46◦47′20′′W), and brought tohe laboratory where they were acclimatized for a week. Therabs were kept in tanks filled with artificial sea water at 20 ppt,ravel, water filter and pieces of brick for the emersion of thenimals. Photoperiod (12:12) was constant as well as the tem-erature (22 ± 3 ◦C). The salinity at 20 ppt was chosen because athis condition the animals are at osmotic equilibrium with outsidenvironment.

.2. Preparation of gill homogenate

Gills filaments of five crabs were separated in anterior and pos-erior parts. For the experiments was used only the anterior gillshat were pooled together, cut with scissors and frozen at −80 ◦Cor posterior use. Gills were homogenized in ice using a homog-nizer (Ika T-10 basic—Ultra Turrax) in 0.05 M Tris–HCl, pH 8.0,ontaining 0.01% Triton X-100. The homogenate was centrifugedwice at 4000 rpm for 15 min, at 4 ◦C, and the supernatant was usedo characterize the ACE.

.3. ACE purification by Sepharose-lisinopril affinityhromatography

ACE from anterior gill homogenate was purified by Sepharose-isinopril affinity chromatography, as previously described [24].he homogenate was desalted in a PD-10 column before the purifi-ation. The Sepharose-lisinopril column was washed with 0.02 MEPES containing 0.3 M NaCl, pH 8.0 and the elution was carriedut with 0.05 M sodium borate, pH 9.5.

.4. Electrophoresis and Western blotting

The Sepharose-lisinopril purified fraction was analyzed by SDS-AGE and Western blotting performed on a 10% polyacrylamideel. The proteins were stained with silver nitrate or transferred to

nitrocellulose membrane (BioRad, CA, USA). For Western blot-ing analysis, the membrane was incubated for 45 min in 0.020 Mris–HCl, pH 7.5, containing 0.15 M NaCl, 0.05% Tween-20 and

mg/mL bovine serum albumin, prior to overnight incubation withnti-angiotensin converting enzyme antibody, clone 9B9 (Milli-ore) diluted 1:500, as determined by Dot Blot protocol.

.5. Synthesis of FRET peptides

The fluorescence resonance energy transfer (FRET) peptidebz-FRQ-EDDnp, which contains the group EDDnp (N-[2-4-initophenyl]-ethylenediamine) attached to a glutamine residue,as synthesized by the solid-phase synthesis method as described

lsewhere [25] using an automated solid-phase peptide synthe-izer Shimadzu model PSSM-8. The FRET peptides, containing thenp group coupled to the �-NH2 of a Lys residue, were synthe-

ized by solid phase methodology as previously described [26].he peptides were purified by semi-preparative HPLC, monitoredy absorbance at 220 nm and by fluorescence emission at 420 nm

ollowing excitation at 320 nm. The molecular mass of purified pep-ides was checked by MALDI-TOF mass spectrometry (TofSpec-E,

icromass). All peptides were stocked in DMSO and the concen-rations measured using the molar extinction coefficient of Dnp

ogical Macromolecules 74 (2015) 304–309 305

and EDDnp (ε365 = 17,300 M−1 cm−1). Angiotensin I and bradykininwere purchased from Sigma-Aldrich Corp. (St Louis, MO, USA).

2.6. Determination of ACE activity in anterior gill homogenate

ACE catalytic activity was determined in anterior gillhomogenates using the FRET peptides Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH [26], Abz-LFK(Dnp)-OH [27] and Abz-FRQ-EDDnp.The hydrolysis was monitored in a Hitachi F-2000 fluorimeterby continuously measuring the fluorescence (�ex = 320 nm and�em = 420 nm) for 5 min. 10–30 �l of the homogenate was incu-bated with 10 �M of the FRET substrates, at 37 ◦C, in 0.1 M Tris–HCl,pH 7.0, containing 0.050 M NaCl and 10 �M ZnCl2, in a final volumeof 0.35 ml. The slope was converted into nanomole of the substratehydrolyzed per minute based on a calibration curve obtained bycomplete hydrolysis of the peptides. We included in the bufferinhibitors that we wanted to suppress the hydrolytic activities(10 �M E64, 1 �M pepstatin, 1 mM PMSF). The specificity of theassay was demonstrated adding 10 �M lisinopril.

2.7. Determination of pH, NaCl and temperature influence oncrab-ACE activity

The pH dependence in crab-ACE hydrolysis was determinedusing 10 �M of Abz-FRK(Dnp)P-OH as substrate in a pH range of5.0–10.5. A four-component buffer comprised of 25 mM glycine,25 mM acetic acid, 25 mM Mes, and 75 mM Tris–HCl [28] was used.The hydrolysis was monitored as described above and velocity ofhydrolysis was determined for each pH value.

To study the influence of NaCl (0–0.5 M) on the catalytic activityof crab-ACE, the enzyme was incubated with 10 �M of Abz-FRK(Dnp)P-OH, at 37 ◦C, in 0.05 M sodium borate (pH 9.5). Thehydrolysis was monitored as described above and the velocity ofhydrolysis was determined for each NaCl concentration.

The effect of temperature (20–55 ◦C) on crab-ACE activity wasdetermined in 0.05 M sodium borate, pH 9.5, using 10 �M of Abz-FRK(Dnp)P-OH as substrate. The enzyme was pre-incubated for5 min at different temperatures and the hydrolysis was monitoredcontinuously at �ex = 320 nm and �em = 420 nm. The velocity ofhydrolysis was determined for each temperature value.

2.8. Determination of crab-ACE activity

The hydrolytic activity of Sepharose-lisinopril purified fraction(crab-ACE) upon the FRET peptides was determined for anterior gillhomogenate. The assays were performed in 0.05 M sodium boratepH 9.5 at 37 ◦C. The Km and Kcat determination for the hydroly-sis of Abz-FRK(Dnp)P-OH by crab-ACE was performed in 0.05 Mborate buffer at pH 9.5. The enzyme concentration for initial ratewas chosen so as to hydrolyze less than 5% of the substrate present.To correct the inner filter effect was used an adjusting equationdetermined experimentally for 0.1–100 �M of Abz-FR-OH [26]. TheKm was calculated from the kinetic parameters by the non-linearregression data analysis Grafit program [29] and the standard devi-ations values was less than 7%.

2.9. Inhibitory effect of lisinopril on crab-ACE

The inhibition of crab-ACE by lisinopril (3–55 nM) was stud-ied using Abz-FRK(Dnp)P-OH as substrate. The assays were carriedout in 0.05 M sodium borate (pH 9.5), at 37 ◦C, after 10 min pre-incubation of the enzyme with the inhibitor. Fluorescence emission

was continuously measured at �ex = 320 nm and �em = 420 nm.Velocities were determined with less than 5% of substrate hydrol-ysis in the absence and in the presence of different inhibitorconcentrations. The IC50 value was obtained by data analysis in

3 of Biological Macromolecules 74 (2015) 304–309

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Fig. 2. Determination of the hydrolytic activity in anterior gill homogenate using

06 P.A. Bersanetti et al. / International Journal

rafit program [29]. The Ki values for the ACE inhibitor were calcu-ated by the following equation:

V0

V1= 1 + [I]

Kiapp

.10. Determination of the cleavage sites

For the determination of the scissile bonds, crab-ACE was incu-ated for 4 h with the natural ACE substrates angiotensin I andradykinin and with the FRET peptides Abz-FRK(Dnp)P-OH, Abz-DK(Dnp)P-OH and Abz-LFK(Dnp)-OH. The assays were performedn 0.05 M sodium borate, pH 9.5, at 37 ◦C. The products were ana-yzed by LC/ESI-MS in a Shimadzu apparatus, model 2010 with aPD-20A Shimadzu UV/vis detector (Tokyo, Japan) and RF-10AXLuorescence detector (Shimadzu, Tokyo, Japan) coupled with anltrasphere C-18 column (5 �m, 4.6 × 250 mm). A linear gradientf 10–80% of solvent B (0.1% TFA in 3:1 acetonitrile/H2O) for 20 minas used after 8 min of isocratic flow of solvent A (0.1% TFA in H2O).

.11. Phylogeny studies

Sequence data were obtained from NCBI (v161) and alignedsing three distinct methods, CLUSTAL v2.1 [30], MUSCLE v3.8.3131] and the alignment using HMMER3 [32] to the ACE domaineptidase M2 (PF01401.13) of Pfam database [33]. The phylogenyf the aligned sequences data was inferred with PhyML [34], withhe HKY85 substitution model. The multiple alignments of the ACEomain was used to infer the phylogenic tree.

. Results

.1. Purification of ACE by Sepharose-lisinopril affinityhromatography

The homogenate obtained from the crab anterior gills wasurified by Sepharose-lisinopril affinity chromatography (960×urification) to apparent homogeneity. Fig. 1 shows the SDS-PAGEnd the Western blot obtained after the purification process. A bandf about 72 kDa was visualized after staining with silver nitrateFig. 1A) or revelation with monoclonal anti-ACE antibody 9B9Fig. 1B).

.2. Activity determination

The ACE catalytic activity in the crude crab homogenateas assayed in 0.1 M Tris–HCl, pH 7.0, containing 0.050 M NaCl

ig. 1. Characterization of crab-ACE purification by Sepharose-lisinopril affinityhromatography. (A) SDS-PAGE (10%) stained with silver nitrate: lane 1, MW stan-ards (Spectra Multicolor Broad Range Protein Ladder–Thermo Scientific); lane 2,nterior gill homogenate; lane 3, purified crab-ACE. (B) Western blotting analysisevealed with anti-ACE antibody.

FRET substrates. The activity for each peptide was considered 1.0 and relative activ-ities after inhibition with lisinopril were calculated. See Material and Methodssection for the hydrolysis conditions.

and 10 �M ZnCl2, with the substrates Abz-FRK(Dnp)P-OH, Abz-LFK(Dnp)-OH, Abz-SDK(Dnp)P-OH and Abz-FRQ-EDDnp (Fig. 2).The anterior crab homogenate could hydrolyze all the tested sub-strates, however, the activity was not totally inhibited by 10 �Mof lisinopril (Fig. 2). This result indicated that, besides ACE, otherproteases present in the anterior gill homogenate are capableto hydrolase the tested peptides. Corroborating this finding, weobserved that the FRET peptide Abz-FRQ-EDDnp, with a blockedC-terminal carboxyl group, was hydrolyzed by a protease differ-ent from ACE, as the action was not inhibited by the selective ACEinhibitor lisinopril.

3.3. Influence of pH and NaCl and temperature in crab-ACEactivity

The effect of pH on the hydrolysis of Abz-FRK(Dnp)-P-OH bycrab-ACE was determined over the pH range of 6.5–10.5 (Fig. 3A).The higher hydrolytic activity was observed at pH 9.5. The influenceof NaCl was also determined using Abz-FRK(Dnp)P-OH as substrate(Fig. 3B). The enzyme reached maximum activity at 75 mM NaCl,however no significant chloride anion activation was detected,even at high salt concentration.

The temperature influence on the hydrolysis of Abz-FRK(Dnp)P-OH by crab-ACE was studied over a range of 20–55 ◦C (Fig. 4). Themaximum activity was reached at around 45 ◦C and after this value,the catalytic activity declined as a result of enzyme thermal dena-turation.

3.4. Determination of crab-ACE activity and cleavage sites

Isolated crab-ACE demonstrated a select activity towards ACEsubstrates (Fig. 5). The enzyme was able to hydrolyze Abz-FRK(Dnp)P-OH with a Km value of 5.5 ± 0.5 �M and kcat value of1.9 ± 0.1 min−1 (Fig. 6). The ACE C-domain selective substrate Abz-LFK(Dnp)-OH was also cleaved by the crab-ACE with a Km value of

−1

6.5 ± 0.3 �M and kcat value of 0.8 ± 0.05 min (data not shown),while the ACE N-domain-substrate Abz-SDK(Dnp)P-OH was resis-tant to hydrolysis. As expected, the peptide Abz-FRQ-EDDnp withthe carboxyl group blocked was not cleaved by crab-ACE.

P.A. Bersanetti et al. / International Journal of Biological Macromolecules 74 (2015) 304–309 307

Fig. 3. pH dependence (A) and NaCl effect (B) on Abz-FRK(Dnp)P-OH hydrolysisby purified crab-ACE. The hydrolysis conditions are described in the Material andM

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4. Discussion

The renin–angiotensin system in invertebrates has beenreviewed [10] and the biological function of this system ranges from

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ethods section.

The cleavage sites determined for natural ACE substrates andRET peptides was analyzed by LC/ESI-MS (Table 1). The crab-ACEas able to hydrolyze Abz-FRK(Dnp)P-OH at the R–K bond and the-domain-specific substrate Abz-LFK(Dnp)-OH at the L–F. The nat-ral ACE substrates angiotensin I (DRVYIHPFHL) was cleaved byrab-ACE only at the F–H bond, generating angiotensin II (m/z 1045)hile bradykinin (RPPGFSPFR) was hydrolyzed at sequential cleav-

ge sites releasing the C-terminal dipeptides F–R and S–P, as seenith ACE [2].

able 1leavage sites of natural and FRET peptides by crab-ACE purified confirmed by LC-S.

Substrate Cleavage site

Bradykinin R-P-P-G-F↓S-P↓F-RAngiotensin I D-R-V-Y-I-H-P-F↓H-LAbz-FRK(Dnp)P-OH Abz-FR↓K(Dnp)P-OHAbz-LFK(Dnp)-OH Abz-L↓FK(Dnp)-OHAbz-SDK(Dnp)P-OH ResistantAbz-FRQ-EDDnp Resistant

Fig. 4. Effect of temperature on Abz-FRK(Dnp)P-OH hydrolysis by purified crab-ACE.The hydrolysis conditions are described in the Materials and Methods section.

3.5. Effect of lisinopril in crab-ACE activity

Using the substrate Abz-FRK(Dnp)P-OH we determined theinhibition constant for the ACE inhibitor lisinopril (Fig. 7). The IC50value determined for crab-ACE was 8.6 ± 0.2 nM and the Ki valuewas 1.26 ± 0.04 nM, indicating that this specific ACE inhibitor is alsovery potent for crab-ACE.

3.6. Phylogenetic inference

The phylogenetic inference results (Fig. 8) obtained based onthe ACE domain region presented a topology, which is consistentwith the genealogy of the taxa. This suggests the orthology of thesequences used for the gene tree.

0

0.2

Fig. 5. Relative activity of the purified crab-ACE using FRET peptides. Maximumactivity was considered 1.0 and relative activity values were calculated. See Materialand Methods section for the hydrolysis conditions.

308 P.A. Bersanetti et al. / International Journal of Biological Macromolecules 74 (2015) 304–309

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Fig. 8. Phylogenetic tree inferred for homologous ACE proteins. The ACE sequencesand accession numbers used for the phylogenetic analysis were: Carcinusmaenas (CAX63316), Pontastacus leptodactylus (CAX48990.1), Locusta migratoria(AAR85358.1), Drosophila melanogaster (AAF53353.2), Strongylocentrotus purpura-tus (XP 798092.2), Xenopus tropicalis (AAI59327.1), Homo sapiens (AAA60612.1),Oryctolagus cuniculus (CAA44428.1), Cynoglossus semilaevis (XP 008312995.1),

he hydrolysis was continuously monitored in a fluorimeter and the data fittedo Michaelis–Menten kinetics. The details are described in Material and Methodsection.

smoregulation, reproduction, memory processes and immuneystem regulation. However, in invertebrates the biologically activeeptides that ACE cleaves still needs to be elucidated. In the presentork, we described the isolation and characterization of a met-

llopeptidase in anterior gills of the crab Ucides cordatus, whichhowed properties similar to mammalian angiotensin I-convertingnzyme (ACE). The purified peptidase, here denominated crab-CE, presented carboxydipeptidyl activity and no endopeptidasectivity upon the tested substrates. The cleavage pattern pre-ented for the hydrolysis of the substrates Abz-FRK(Dnp)-P-OH,bz-LFK(Dnp)-OH, angiotensin I and bradykinin is identical to thatbserved for the mammalian ACE. Another characteristic observeds that crab-ACE is not capable to hydrolyze peptides with blockedarboxyl group, as observed for the peptide Abz-FRQ-EDDnp.

The SDS-PAGE and Western blotting analysis showed the

resence of a single domain enzyme in crab-ACE with about2 KDa. Other characteristic of crab-ACE similar to the mammalian

soform is the Km value determined for the hydrolysis of the Abz-RK(Dnp)P-OH (5.5 �M). This value is close to the Km value of

Concentration of lisinopril (nM)

0 10 20 30 40 50 60

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(%)

0

20

40

60

80

100

ig. 7. Purified crab-ACE inhibition by lisinopril. The inhibitory activity was deter-ined by measuring the remaining hydrolytic activity after pre-incubation (10 min)

f active enzyme with different inhibitor concentrations. The enzyme–inhibitorixture was added to a solution containing Abz-FRK(Dnp)P-OH in the same buffer.

Alligator mississippiensis (XP 006271633.1), Gallus gallus (ETE62480.1), Hydra vul-garis (XP 004208490.1), Theromyzon tessulatum (AAS57725), Crassostrea gigas(EKC26119.1).

4.0 �M described for the hydrolysis of the same FRET peptide byrecombinant wild-type ACE [26]. The NaCl effect on crab-ACE activ-ity was also very similar to the profile showed by recombinantwild-type ACE [26] as well as the inhibition by the lisinopril.

Curiously, crab-ACE is different from humans and other mam-malian isoforms, in the pH profile, with maximum activity at pH 9.5.This optimum pH suggests that an amino acid residue in catalyticsite must be deprotonated for optimal activity of the crab-ACE.Corroborating this result, the substrate Abz-SDK(Dnp)P-OH, havingan acid residue in the S1 subsite [35], was not hydrolyzed by thecrab-ACE. An ACE isoform from mollusk Mytilus edulis hydrolyzedangiotensin I at optimum pH of about 8.4 [36]. In addition, Lipkeet al. showed that an ACE isoform present in fish is active overa broad range of pH with an optimum activity on the substratehippuryl-l-histidyl-l-leucine (HHL) at pH 10.5 [37]. These authorssuggested that poikilothermic animals have blood pH that varieswith changes in environmental temperature. Therefore, aquaticorganisms that do not maintain constant body temperature, suchas crustaceans, should have enzymes that function well over a widepH range, as observed here.

Crab-ACE maximum activity was achieved at 45 ◦C, decreasingat higher temperatures. Takada et al. showed that kidney ACEof different mammalian species is stable at 40 ◦C, with posteriorinactivation [38]. O’Neill et al., using site-directed mutagenesis toreduce N-glycans contents in C- and N-domain of ACE, demon-strated a decrease in thermal stability, suggesting that certain

N-glycans have an important effect on the thermodynamic prop-erties of the enzyme. This authors also demonstrated that the ACEN-domain is more thermostable than the C-domain, probably dueto the greater number of glycosylation sites [39]. Considering the

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P.A. Bersanetti et al. / International Journal

rab- ACE thermal stability, we can speculate that the enzymeresents a low level of glycosylation, which is consistent with the-domain characteristic.

We found a molecular mass for crab-ACE of around 72 kDa,uggesting that it is a single domain enzyme. Several ACE iso-orms present in invertebrates are described as containing onlyne domain. Laurent and Salzet [11] described an ACE isoformn brain of leech as a single domain enzyme. Other mollusks alsoontain ACE as a single domain [36] in pedal ganglia and in theserganisms this enzyme is implicated in the metabolism of severaleuropeptides. Most insects have single domain enzyme [40,41]xcept for Anopheles, having an ACE with two domain structure19]. A two-domain enzyme was also characterized in crustaceansepatopancreas [15], suggesting that the evolution of this enzyme

s far from complete.The results obtained from the alignment of the active sites of

CE from different species demonstrated that the representativerganisms from the Phylum Arthropoda (Carcinus maenas, Pontas-acus leptodactylus, Locusta migratoria and Drosophila melanogaster)re phylogenetically close as shown in the phylogenetic tree. Asxpected, the vertebrate cluster of ACE isoforms is distinct from therthropod cluster. The presence of ACE with two domains in variouspecies suggests that the gene duplication may have occurred veryarly in evolution, as a common ancestor of Arthropoda, Cnidariand Chordata appeared 700 million years ago. It is still possible thathe ACE gene duplication occurred more than once in evolutionaryime frame [42].

. Conclusion

We purified and characterized an ACE isoform (crab-ACE) fromnterior gills of Ucides cordatus that presents many properties sim-lar, but temperature and pH activities were very different from

ammalian ACE. Further studies are necessary to elucidate the nat-ral substrates and the physiological function of this ACE isoform

n the gills of these invertebrates.

cknowledgments

This work was funded by Fundac ão de Amparo a Pesquisa dostado de São Paulo (FAPESP) and Conselho Nacional de Desen-olvimento Científico e Tecnológico (CNPq). We acknowledge Dr.dward D. Sturrock for the generous gift of the Sepharose-lisinoprilesin.

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