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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 84-88, January 1992 Biochemistry A --inactivating endopeptidase in Xenopus laevis skin secretion (metailoendopeptidase/neutral endopeptidase/) KRISHNAMURTI DE MORAIS CARVALHO*, CARINE JOUDIOU, HAMADI BOUSSETTA, ANNE-MARIE LESENEY, AND PAUL COHEN Groupe de Neurobiochimie Cellulaire et Moldculaire de l'Universitd Pierre et Marie Curie, Unit6 de Recherche Associ6e 554 au Centre National de la Recherche Scientifique, % Boulevard Raspail, 75006 Paris, France Communicated by I. Robert Lehman, September 16, 1991

ABSTRACT An endopeptidase was isolated from Xenopus Indeed the Ser-Phe , or a related motif such as laevis skin secretions. This enzyme, which has an apparent Phe-Phe, Ala-Phe, or His-Phe, is often present near the molecular mass of 100 kDa, performs a selective cleavage at the carboxyl terminus of substances from the and Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, tachykinin families (1). Xaa-Phe, Xaa-Leu, or Xaa-Ile was or Gly) of a number of peptide , including atrial also found frequently at a similar position in other peptide natriuretic factor, , H, , hormone sequences of higher organisms, notably in atrial , neuromedins B and C, and litorin. The peptidase natriuretic factor (ANF). exhibited optimal activity at pH 7.5 and aKm in the micromolar We have purified this enzyme 2029-fold and demonstrate range. No cleavage was produced in , ocytocin, that it inactivates ANF by exclusive cleavage of the Ser25- minigastrin I, and [Leu5Jenkephalin, which include in their Phe26 bond and similarly inactivates a number of important sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The en- peptide hormones such as somatostatin, bradykinin, neuro- dopeptidase activity was inhibited by divalent cation chelators medins B and C, litorin, substance P, and angiotensin II. and by only at high concentrations (ICss = 50 jAM), whereas it was insensitive to classical inhibitors of AND chymotrypsin, angiotensin convertase, and serine and cysteine MATERIALS METHODS peptidases, as well as carboxypeptidases. It is hypothesized that Purification Procedure. Exudates were obtained from three this enzyme, which is distinct from neutral endopeptidase (EC X. laevis (Centre National de la Recherche Scientifique, 3.4.24.11), constitutes the prototype of a family of related Montpellier, France), and submitted to Sephadex G-50, metalloendopeptidases that inactivate peptide substrates by DEAE-Sephadex, and aminoethylagarose hydrophobic ab- cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-fle bond. sorption chromatography as in ref. 3. The fractions contain- ing endopeptidase activity were then applied to a Nucleosil A rather limited number ofpeptidases seem to be involved in C4 300-A (8 x 75 mm) HPLC column (SFCC-Shandon, the postsecretory inactivation of messen- Eragny, France) and eluted with 10 mM sodium phosphate/ gers. The importance of these proteolytic mechanisms in 1.5 M sodium chloride buffer, pH 7.4, at a flow rate of 0.5 regulating hormonal action can be demonstrated by the fact ml-min-1, for 10 min followed by a linear gradient from 1.5 to that their effects can be prolonged in vivo or in vitro by 0 M sodium chloride/10 mM sodium phosphate buffer, pH selective inhibitors ofthese enzymes. This has been shown in 7.4, in 10 min. Subsequently, a gradient from 0% to 30% the case of the -degrading enzyme neutral en- (vol/vol) in 10 mM sodium phosphate buffer, pH dopeptidase (NEP; enkephalinase, EC 3.4.24.11) and in the 7.4, in 60 min eluted the enzyme, which was recovered at 25% case of angiotensin-converting enzyme (ACE; EC 3.4.15.1). methanol. The purified enzyme was resistant to 30%o meth- Many of these previously identified peptidases cleave with a anol and 1.5 M sodium chloride (90%o of initial activity limited selectivity and sometimes at multiple sites. remained; data not shown). The endopeptidase was com- The skin secretions of Xenopus laevis contain an extraor- pletely separated from the copurifying RXVRG-endopro- dinary number of peptide hormones eliciting a broad spec- tease activity (eluted with 12% methanol). The active frac- trum of biological actions (reviewed in ref. 1). In addition to tions were pooled, concentrated to a 30-,ul final volume, and those substances so far identified exclusively in the batra- analyzed by polyacrylamide gel electrophoresis under either chian skin exudate such as caeruleins, magainins, levitide, nondenaturing or denaturating conditions using the PhastGel PGLa, xenopsin, etc. (1, 2), the secretory glands of these system (Pharmacia) (3, 4). content was evaluated by amphibians can also produce mammalian peptide hormones the method of Bradford (5). and some of their analogs, such as substance P, corti- Enzyme Assays. Endopeptidase activity was monitored as in coliberin, thyroliberin, and angiotensin I. In the course of ref. 3, using as substrate the diaminobenzylthiocyanate isolating the enzyme responsible for the activation of some X. (DABTC) derivative of [DArg8]kermit, a derivative of kermit laevis skin secretion hormone precursors by cleavage at the (Asp-Val-Asp-Glu-Arg-Asp-Val-Arg-Gly-Phe-Ala-Ser-Phe- Arg-Gly bond of a consensus Arg-Xaa-Val-Arg-Gly (RX- Leu-NH2) that undergoes a single cleavage at the Ser12-Phe13 VRG) sequence, we have detected a contaminating proteo- bond. When other peptide hormones were used as substrates, lytic activity (3). This endopeptidase copurifies with the the conditions were as follows: 1-2 nmol of peptide incubated RXVRG-endoprotease of X. laevis skin secretions and cleaves at the Ser-Phe bond situated on the carboxyl side of Abbreviations: NEP, neutral endopeptidase (EC 3.4.24.11); ACE, the RXVRG consensus sequence in a synthetic peptide (3). angiotensin converting enzyme (EC 3.4.15.1); PHIE, peptide- hormone-inactivating endopeptidase; ANF, atrial natriuretic factor; DABTC, diaminobenzylthiocyanate. The publication costs of this article were defrayed in part by page charge *On leave from: Depto de Fisiologia e Farmacologia da Universidade payment. This article must therefore be hereby marked "advertisement" Federal do CearA (UFC), CX Postal 657, Rua Cel Nunes de Melo in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1127, 60.000-Fortaleza, CE, Brazil. 84 Downloaded by guest on September 27, 2021 Biochemistry: Carvalho et al. Proc. Natl. Acad. Sci. USA 89 (1992) 85

1-5 hr in 100 mM sodium phosphate buffer, pH 7.4, in the Phe-Phe, His-Phe, Gly-Leu, His-Leu, or Tyr-Ile bond of the presence of 10 ng ofpure enzyme in 20 pkl. After heating 10 min tested . Hydrolysis of the Ser25-Phe26 bond of ANF- at 1000C, the resulting products were separated by HPLC (5-28) removed the Phe26-Arg27-Tyr28 tail and led using a Nucleosil 5-,um C18 column (146 x 4.5 mm) eluted as to stoichiometric amounts of both the NH2-terminal 5-25 described in the legend of Fig. 2. Substrate and product(s) fragment and this tripeptide (fragments a and b, Fig. 2A). For were monitored by UV absorbance at 220 nm and identified by substance P, the major hydrolytic process occurred at the analysis using a PicoTag station (Waters) or by Gly9-Leu10 bond (fragments a and c, Fig. 2B) and a minor one amino-terminal sequencing (6). Inhibitors were tested at var- at the Phe7-Phe8 doublet (fragments b and d, Fig. 2B). In the ious concentrations (Table 2), using the routine conditions of case ofangiotensin II only one cleavage was produced, at the the endopeptidase assay (see above) and either ANF-(5-28) or Tyr4-Ile5 bond (fragments a and b, Fig. 2C). Bradykinin was [DArg8]kermit as substrate. Inhibitions were expressed as cleaved at the Gly4-Phe5 bond (fragments a and d, Fig. 2D) percentages of the reference activity measured in the absence with a minor cleavage at the Pro7-Phe8 bond (fragments b and of chemical reagents under the same experimental conditions. c, Fig. 2D). Somatostatin-14 undergoes a single cleavage at The pH profile ofendoprotease activity was obtained by using the Phe6-Phe7 bond situated in the disulfide-linked loop ofthe either [DArg8]kermit or ANF-(5-28) as substrate (2 nmol per tetradecapeptide. This resulted in a single product (Fig. 2E) assay) incubated 1 hr with 10 ng of pure enzyme in a 0.1 M (fragment a was identified by NH2-terminal sequencing). In sodium phosphate buffer adjusted to cover a pH range of 5.8 neuromedin C, as well as in and litorin (data to 8.0 (Fig. 4). Under these conditions enzyme kinetics re- not shown), a major hydrolysis was observed at the His8- mained linear, indicating that the endopeptidase was stable at Leu9 or His8-Phe9 bond (fragments a and d, Fig. 2F) and a the pH values used. Enzyme and inhibition assays were run on minor cleavage at Gly7-His8 (fragments b and c, Fig. 2F). average two or three times for each peptide. Analysis of The pure enzyme exhibited high affinity toward all the products was performed at least in duplicate. cleaved substrates. Km values in the range of 18-63 ,M were Peptides and Chemicals. [DArg8]kermit, ANF-(24-28), measured toward ANF-(5-28), substance P, somatostatin-14, [Ala2IANF-(24-28), and [His25]ANF-(24-28) were prepared by bradykinin, neuromedin C, and angiotensin II (Fig. 3). solid-phase synthesis (7, 8) on a Multisynthesizer NPS 4000 Other peptides containing a Xaa-Phe, Xaa-Leu, or Xaa-Ile (Neosystem, Strasbourg, France), purified by HPLC, and motif, including ocytocin, vasopressin, minigastrin I, checked as described (4, 9). Chemicals and peptide substrates [Leu5]enkephalin, and its amidated derivative were not cleaved or fragments were from Neosystem or Sigma. (Fig. 3). Therefore, pentapeptides reproducing or mimicking the COOH-terminal sequence of ANF(24-28) were used (Fig. 3). In all cases they were not cleaved at the Xaa-Phe bond but RESULTS appeared to be inhibitors of ANF cleavage by the endopepti- Purification of X. laevis Endopeptidase. Complete purifica- dase. [Leu5]Enkephalin behaved as an inhibitor of the ANF tion of peptide-hormone-inactivating endopeptidase (PHIE) Ser5-Phe'2 bond hydrolysis (IC50 = 54 ,uM). was achieved by using four successive steps, with a final The endopeptidase of X. laevis secretions exhibits a pref- enrichment factor of 2029 (Table 1). This factor is probably erence for cleavage on the NH2-terminal side of Phe, Leu, or underestimated, since at the early steps ofthe purification the Ile in an Xaa-Phe, Xaa-Leu, or Xaa-Ile dipeptide. This Ser-Phe cleavage activity is obscured by contaminating pro- activity is governed by other structural parameters such as teases. The high yield could be attributed to the presence of the size ofthe substrate and probably its conformation. When endogenous inhibitor(s) (3), to the multiple proteolytic activ- at the COOH terminus ofthe peptide, the Xaa-Phe, Xaa-Leu, ities present in the exudate, or to both. These proteases might or Xaa-Ile motif was not cleaved as in the case of minigastrin degrade the substrate or the endopeptidase products in the I and angiotensin II (Fig. 3). When the Xaa-Phe, Xaa-Ile, or earliest step of the purification, leading to an underestimate Xaa-Leu motif was at positions +2 in preprothyroliberin- of the starting specific activity. The activity was enzymati- (160-169) (10), +3 in ANF-(24-28) fragment and its deriva- cally homogeneous with respect to the Ser-Phe cleavage tives, and ocytocin, vasopressin, and neuromedin B, or +4 in produced on [DArg8]kermit and ANF-(5-28) or the Gly9- [Leu5]enkephalin, [Leu ]enkephalinamide, and the pen- Leu10 bond cleavage of substance P. The peptidolytic activ- tapeptides derived from ANF from the NH2 terminus in a ities monitored by using three substrates were eluted per- number ofpeptides (Fig. 3 Lower) no cleavage was observed. fectly together and coincided with a discrete protein peak This indicates that the position of the conserved motif (Fig. 1A). The purity of the enzyme was demonstrated by gel (Xaa-Phe, Xaa-Leu, or Xaa-Ile) within the peptide hormone electrophoresis under both nondenaturing and denaturing substrate is an important and limiting factor. Thus, a peptide conditions. In the absence ofSDS, the activity coincided with containing Phe, Leu, or Ile with four amino acids on the a single protein band (Fig. 1B); in its presence, the protein NH2-terminal and 1 amino acid on the COOH-terminal side migrated as a single band, indicating an apparent molecular appears to be a minimal requisite for hydrolysis by PHIE. mass of 100 kDa (Fig. 1C). The enzyme showed a high Optimal pH and Inhibitor Prorfles. Optimal pH was 7.5 stability over periods of 24 hr or more in the routine assay. (Fig. 4B). EDTA, EGTA, o-, or dithiothreitol Substrate Selectivity. The X. laevis PHIE performed in in the millimolar concentration range significantly inhibited most cases a single or major cleavage at a Ser-Phe, Gly-Phe, the activity, suggesting a metalloendopeptidase character of Table 1. Purification of the X. laevis PHIE Total protein, Total activity, Specific activity, Purification, Yield, Step mg nmol/hr nmol/hr per mg fold % Skin exudate 285 1549 5.4 1 100 Sephadex G-50 194 1394 7.2 1.3 90 DEAE-Sephadex 57 1043 18.3 3.4 67 Aminoethylagarose 1.8 660 367 68 43 C4-silica HPLC 0.023 252 10,956 2029 16 The enzyme activity was evaluated in a 20-Al aliquot from each purification step; [DArg8]kermit was used as the substrate. Activities were expressed as nmol of peptide cleaved at the Ser-Phe bond per hr and protein content was evaluated as in ref. 5. Specific activity is expressed as nmol of substrate cleaved per hr per mg of protein. Downloaded by guest on September 27, 2021 86 Biochemistry: Carvalho et al. Proc. Natl. Acad. Sci. USA 89 (1992)

A |2000 0-

0 6 CD ->~OL 11 > W (n ';- N 1000 m o Fa N > = CAj N

-O.a)Ca- 0 C2) C,)

time. min E O SOM C: D BK E F NC

B 100 d

so a C 0 b C 50 c C0

0 5 10 15 0 5 10 15 0 5 10 15 co Retention time, min FIG. 2. Elution profile of fragments of various hormone sub- strates generated by the action of the X. laevis PHIE. (A) ANF. (B) Substance P. (C) Angiotensin II. (D) Bradykinin. (E) Somatostatin- 14. (F) Neuromedin C. After incubation of each peptide (1-2 nmol) in the presence of pure enzyme (10 ng), the reaction was stopped by heating for 10 min at 100TC and the entire sample was injected into the HPLC column (Nucleosil 5-gum, C18, 146 x 4.5 mm) and eluted a 9 18 27 36 with a 0-45% gradient of acetonitrile in 25 min. Fragments were Fraction number identified by amino acid composition, by reference to a standard, and by NH2-terminal sequencing. C the enzyme (Table 2). In contrast, classical inhibitors of either NEP or ACE were either poorly efficient or inefficient kDa up to micromolar and millimolar concentrations, respective- ly-i.e., at concentrations well above those reported to be 94 fully efficient in inhibiting these endopeptidases (see Discus- 67 sion). Phosphoramidon partially inhibited the enzyme at concentrations above 0.1 AtM (IC50 of 50 AM). Total inhibi- 43 tion of the enzyme was not reached at concentrations as high

30 DABTC-[DArg8]kermit (0, 0-80 scale), ANF-(5-28) (a, 0-2000 20.1 scale), and substance P (o, 0-2000 scale). The arrows indicate the fractions containing the RXVRG endoprotease activity (3). (B) 14.4 Electrophoresis was on an 8-25% polyacrylamide gradient gel of the PhastGel system (Pharmacia). Aliquots of the enzyme from the final purification step, containing 0.5 ,Ag of protein, were loaded sepa- rately on two parallel lanes. After migration, one lane was silver stained and the other was cut into 1-mm slices. Each slice was incubated with 50 ul of 0.1 M sodium phosphate buffer, pH 7.4, FIG. 1. Purification of the X. laevis PHIE. (A) The enzyme containing 50 pmol of DABTC-[DArg8]kermit for 2 hr at 37TC. recovered from the aminoethylagarose absorption step was submit- Enzyme activity was expressed as nmol of substrate cleaved at the ted to reverse-phase HPLC on a C4-silica 300-A HPLC column Ser12-Phe"3 bond per hr. (C) SDS/2-mercaptoethanol polyacrylam- equilibrated with 10 mM sodium phosphate/1.5 M sodium chloride ide gel electrophoresis of X. laevis PHIE recovered from the final buffer, pH 7.4, at room temperature and run at a flow rate of 0.5 purification step (see A). An aliquot ofthe active fractions recovered ml-min-. Elution with this buffer continued for 10 min and was on the C4-silica HPLC step was submitted to analysis on an 8-25% followed by a linear gradient from 1.5 to 0 M sodium chloride in 10 polyacrylamide gradient gel in the presence of SDS/2-mercapto- mM sodium phosphate buffer, pH 7.4 in 10 min. Subsequently, a ethanol in the PhastGel system. were visualized by silver gradient from 0o to 30%o methanol in 10 mM sodium phosphate staining. Left lane, molecular mass markers; from top to bottom: buffer, pH 7.4, in 60 min eluted the enzyme. Absorbance was phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbu- monitored at 220 nm (-); aufs, absorbance units at full scale. min (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 One-milliliter fractions were collected and 20-ul aliquots were as- kDa), and a-lactalbumin (14.4 kDa). Right lane, 0.5 ,ug ofthe enzyme sayed for enzymatic activity. Three different substrates were used: recovered from the previous C4-silica HPLC purification step (A). Downloaded by guest on September 27, 2021 Biochemistry: Carvalho et al. Proc. Natl. Acad. Sci. USA 89 (1992) 87 Km IC50 (1O-5M) (1-5M)

(5-28) ANF SSCFGGHMDRIGAQSGLGC N SIF R Y 5 Substance P R P K P Q N F'F GfL M(NH2) 2 Angiotensin II D R V YfI H P F 6.3 Bradykinin R P P G$F S P'F R 4.1 Somatostatin-14 A G C K N Ff W K T F T S C 1.8 Neuromedin C G N H W A V GiHIL M (NH2) 2.2 Litorin pE Q W A V G HfF M(NH2) 2.4 Neuromedin B G N L W A T G4HfF M(NH2) 2.1

Minigastrn I L EEEEE A Y G W M D F (NH2) Angiotensin II D R V Y I H P F FIG. 3. Amino acid sequences of the peptides used as substrates (160-169)Preprothyroliberin S F P W M E S D V T for the X. Iaevis PHIE. Large ar- (24-28) ANF N S F R Y 20 rows indicate the major cleavage [Ala25] (24-28) ANF N A F R Y - observed; smaller arrows corre- spond to minor cleavage sites. The [His25] (24-28)ANF N H F R Y sequences were aligned to high- Vasopressin C Y F Q N C P R G (NH2) - - light the Xaa-Phe, Xaa-Leu, and OcUcin C Y I Q N C P L G(NH2) Xaa-Ile bonds. Km values were measured by using five different Neuromedin B G N LW A T G H F M(NH2) concentrations of substrate under [Leu5] Enkephalin Y G G F L - 5.4 conditions where a maximum of [Leu5] Enkephalinamide Y G G F L (NH2) - 3.5 5% cleavage was measured. as 100 AM (Fig. 4A). Thiorphan and related NEP inhibitors inactivating enzymes distinct from NEP and ACE, a hypoth- exerted some effect only for concentrations above 1 ,M and esis supported by a set of observations. inhibitors for cysteine, serine, and carboxyl proteases had no First, this dipeptide is a site of selective and inactivating effect up to 1 mM (Table 2). cleavage in -containing systems. ANF under- goes selective removal of the Phe26-Tyr28 tripeptide tail by cleavage at the Ser-Phe bond in a number oftissues, including DISCUSSION vascular smooth muscle endothelial cells (13, 14), Clearance of peptide hormone by proteolytic inactivation is neuroblastoma NB-OK-1 cells (15), and bovine (16). a general mechanism that can be topographically associated The enzyme responsible for this truncation has not been, as with receptors in the target organs (11, 12). The enzyme yet, either isolated or clearly characterized, but it might be a presently described produces a major, selective cleavage at metalloendopeptidase (14-17). Similarly, cleavage at the an Xaa-Phe, Xaa-Ile, or Xaa-Leu in a number Phe6-Phe7 site of somatostatin has been reported (18, 19). of peptides that have in common the presence of these Concerning substance P, various cleavages, including Phe- in their sequence. Since this enzyme cleaves the Phe bond hydrolysis, were observed in multiple target cells, Xaa-Phe bond of ANF, somatostatin, and substance P, for including synaptic membranes of the substantia nigra, rat example, which are known to be inactivated by these trun- brain membranes, and human cerebrospinal fluid (20-24). cations, we suggest that it is a prototype of a class of Second, it is clear that the X. laevis PHIE is distinct from the NEP, ACE, the substance P-degrading enzyme (EC 3.4.24.-), and other comparable activities (22-24). ANF can be degraded in vitro by NEP, which cleaves, essentially, and in a rate-limiting way, at the Cys7-Phe8 bond (11, 25), whereas only a secondary, and slower reaction was observed at the Ser25-Phe26 bond (11, 25, 26). Moreover, the skin secretion .520-CO enzyme, which does not cleave enkephalin, was sensitive to phosphoramidon, and other classical NEP inhibitors, only at 0) concentrations 100- to 1000-fold higher than those operating on the NEP (27). In vivo studies have demonstrated that a) degradation of the endogenous pool of ANF, in the kidney, could not be prevented by NEP inhibitors, suggesting the involvement ofanother enzyme resistant to phosphoramidon up to micromolar concentrations (11, 12). Finally, the X. laevis PHIE was insensitive to captopril up to millimolar 9 8 7 6 5 4 5 6 7 8 9 concentrations, indicating clearly a different enzyme from -log[phosphoramidon] (M) pH ACE. Moreover the latter, as well as NEP, tolerates shorter peptide substrates than the skin secretion enzyme (28). With FIG. 4. pH and phosphoramidon inhibition profiles of the X. to substance from the Iaevis PHIE. The activity was measured in a standard assay (2 nmol respect P-degrading endopeptidase of substrate-i.e., [DArg8]kermit or ANF-(5-28)-in the presence of brain it is clear that X. laevis PHIE promotes different 10 ng of pure enzyme in 0.1 M sodium phosphate buffer in a final cleavages than those produced by the rat enzyme (Pro-Gln, volume of 20 Aul for 1 hr at 37°C). The buffer was adjusted to pH 7.5 Gln-Gln, and Gln-Phe hydrolysis) or by the human species (A) or to cover the pH range of 5.8-8.0 (B). (Gln-Phe, Phe-Phe, and Phe-Gly) (20, 22). Downloaded by guest on September 27, 2021 88 Biochemistry: Carvalho et al. Proc. Natl. Acad. Sci. USA 89 (1992) Table 2. Effects of protease inhibitors on the X. laevis by the Ministere de l'Education Nationale de la Jeunesse et des PHIE activity Sports, by the Centre National de la Recherche Scientifique (URA 554), the Fondation pour la Recherche Medicale, the Ligue Nationale Peptidase Conc., Inhibition, Franqaise Contre le Cancer, the Laboratoire d'Ingdnierie des Pro- class Inhibitor mM % teines of the Commissariat a l'Energie Atomique de Saclay, the Metallo o-Phenanthroline 1 98 Association Franqaise Contre les Myopathies (AFM), and the Comissao de Aperfeiqoamento de Pessoal de Nfvel Superior 0.1 5 (CAPES), Brazil. EDTA 10 67 1 59 1. Bevins, C. L. & Zasloff, M. (1990) Annu. Rev. Biochem. 59, EGTA 10 63 395-414. 1 51 2. Gibson, B. W., Poulter, L., Williams, D. H. & Maggio, J. E. Dithiothreitol 10 66 (1986) J. Biol. Chem. 261, 5341-5349. 1 49 3. Kuks, P. F. M., Creminon, C., Leseney, A. M., Bourdais, J., Cysteine PCMPS 1 0 Morel, A. & Cohen, P. (1989) J. Biol. Chem. 264, 14609-14612. 4. Plevrakis, I., Clamagirand, C., Crdminon, C., Brakch, N., Iodacetamide 1 1 Rholam, M. & Cohen, P. (1989) Biochemistry 28, 2705-2710. NEM 1 0 5. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Serine PMSF 1 0 6. Chang, J. Y. (1983) Methods Enzymol. 91, 455-466. STI 1 0 7. Tam, J. P., Heath, W. H. & Merrifield, R. B. (1983) J. Am. Aprotinin 1 0 Chem. Soc. 105, 6442-6455. TPCK 1 2 8. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154. Carboxy Pepstatin 1 0 9. Nicolas, P., Delfour, A., Boussetta, H., Morel, A., Rholam, M. & Cohen, P. (1986) Biochem. Biophys. Res. Commun. 140, GEMSA 0.1 1 565-573. NEP Phosphoramidon 0.001 41 10. Bulant, M., Delfour, A., Vaudry, H. & Nicolas, P. (1988) J. 0.005 52 Biol. Chem. 263, 17189-171%. Acetorphan 0.001 15 11. Roques, B. P. & Beaumont, A. (1990) Trends Pharmacol. Sci. 0.005 26 11, 245-249. Kelatorphan 0.001 12 12. Schwartz, J. C., Gros, C., Lecomte, J. M. & Bralet, J. (1990) 0.005 20 Life Sci. 47, 1279-1297. 13. Johnson, G. R., Arik, L. & Foster, C. J. (1989) J. Biol. Chem. Retrothiorphan 0.001 1 264, 11637-11642. Thiorphan 0.001 0 14. Johnson, G. R. & Foster, C. J. (1990) Biochem. Biophys. Res. ACE Captopril 1 0 Commun. 167, 110-116. Aspecific Benzamidine 0.1 0 15. Delporte, C., Poloczek, P., Gossen, D., Tastenoy, M., Winand, TAME 1 0 J. & Christophe, J. (1991) Eur. J. Pharmacol. 207, 81-88. 16. Toll, L., Brandt, S. R., Olsen, C. M., Judd, A. K. & Almquist, EnzymeactivitywasassayedwitheitherANF-(5-28)or[DoAigkermit R. G. (1991) Biochem. Biophys. Res. Commun. 175, 886-893. in the presence ofa single inhibitor at a time. PCMPS, p-(chloromer- 17. Rugg, E. L., Aiton, J. F. & Cramb, G. (1988) Biochem. Bio- curi)benzenesulfonic acid; PMSF, phenylmethanesulfonyl fluoride; phys. Res. Commun. 152, 294-300. TAME, tosylarginylmethyl ester; STI, soybean trypsin inhibitor; 18. Weber, M., Cole, T. & Conlon, J. M. (1986) Am. J. Phys. 250, TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; NEM, N-eth- 679-685. ylmaleimide; GEMSA, guanidylethylmercaptosuccinic acid. 19. Harmar, A. J., Pierotti, A. R. & Lightman, S. L. (1986) in Neuroendocrinology, eds. Lightman, S. L. & Everitt, B. J. In conclusion, the X. laevis enzyme is a metalloendopep- (Blackwell, Oxford), pp. 389-408. tidase that hydrolyzes a peptide bond in a dipeptide motif 20. Endo, S., Yokosawa, H. & Ishii, S. (1988) J. Biochem. 104, containing a Phe, Leu, or Ile residue on the carboxyl side, 999-1006. thus exhibiting thermolysin-like activity (27, 28). Moreover, 21. Nyberg, F., Greves, P. L., Sundgvist, C. & Terenius, L. (1989) PHIE cleaves only this type of bond efficiently when in- Biochem. Biophys. Res. Commun. 125, 244-250. cluded in a sequence containing more than five residues, an 22. Lee, C., Sandberg, B. E. B., Hanley, M. R. & Iversen, L. (1981) Eur. J. Biochem. 114, 315-327. additional and noticeable difference from the structural req- 23. Stephenson, S. L. & Kenny, A. J. (1988) Biochem. J. 255, uisites for NEP activity (28). Such an enzyme, or a closely 45-51. related one, could be present in mammalian tissues, such as 24. Oblin, A., Danse, M. J. & Zivkovic, B. (1989) Life Sci. 44, those containing ANF receptors (15, 16). Isolation of com- 1467-1474. parable activities present in kidney or neural cells (14-16) 25. Bourne, A. & Kenny, A. J. (1990) Biochem. J. 271, 381-385. may allow for a precise comparison. 26. Olins, G. M., Krieter, P. A., Trapani, A. J., Spear, K. L. & The physiological function of this metalloendopeptidase is Bovy, P. R. (1989) Mol. Cell. Endocrinol. 61, 201-208. 27. Almenoff, J. & Orlowski, M. (1984) J. Neurochem. 42 (1), not known, but it might be related to the inactivation of 151-157. numerous peptides of the angiotensin, bombesin, and tachy- 28. Pozsgay, M., Michaud, C. & Orlowski, M. (1985) Biochem. families, present in the skin secretions ofX. laevis (1). Soc. Trans. 13, 44-50. Finally, PHIE constitutes the sixth peptide hormone metab- 29. Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. & Matsuo, olism-related enzyme activity that has been described so far H. (1986) Biochem. Biophys. Res. Commun. 137, 984-991. in X. laevis skin secretions (3, 29-32). Elucidation of its 30. Mollay, C., Vilas, U., Hutticher, A. & Kreil, G. (1986) Eur. J. primary structure will indicate if the signature (i.e., the Biochem. 160, 31-35. of a number of 31. Darby, N. J., Lackey, D. B. & Smyth, D. G. (1991) Eur. J. consensus His-Glu-Xaa-Xaa-His sequence) Biochem. 195, 65-70. related thermolysin-like Zn2+-metalloendoproteases (28, 33), 32. Resnick, N. M., Maloy, L. W., Guy, H. R. & Zasloff, M. including NEP (34), is present in its sequence. (1991) Cell 66, 541-554. 33. Jongeneel, C. V., Bouvier, J. & Bairoch, A. (1989) FEBS Lett. The help of 0. Le Blanche, P. F. M. Kuks, and Dr. A. Pierotti is 242, 211-214. greatly appreciated. We are grateful to Ch. Fahy, N. Barre, and Ch. 34. Devault, A., Lazure, C., Nault, C., Le Moual, H., Seidah, Creminon for excellent help and to Dr. P. Fromageot for his N. G., Chretien, M., Kahn, P., Powel, J., Mallet, J., Beau- continuous interest. Drs. B. Roques and M. Cl. Fournie Zaluski mont, A., Roques, B. P., Crine, P. & Boileau, G. (1987) EMBO kindly provided the NEP inhibitors. This work was supported in part J. 6, 1317-1322. Downloaded by guest on September 27, 2021