©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing of the Precursors to Neurotensin and Other Bioactive Peptides by Cathepsin E (*)

(Received for publication, March 14, 1995; and in revised form, June 7, 1995)

Takashi Kageyama (1)(§),   Masao Ichinose (2) Satoshi Yonezawa (3)

From the (1)Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484, Japan, the (2)First Department of Internal Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, and the (3)Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-03, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cathepsin E (EC 3.4.23.34), an intracellular aspartic proteinase, was purified from monkey intestine by simple procedures that included affinity chromatography and fast protein liquid chromatography. Cathepsin E was very active at weakly acidic pH in the processing of chemically synthesized precursors such as the precursor to neurotensin/neuromedin, proopiomelanocortin, the precursor to xenopsin, and angiotensinogen. The processing sites were adjacent to a dibasic motif in the former two precursors and at hydrophobic recognition sites in the latter two. The common structural features that specified the processing sites were found in the carboxyl-terminal sequences of the active peptide moieties of these precursors; namely, the sequence Pro-Xaa-X`aa-hydrophobic amino acid was found at positions P4 through P1. Pro at the P4 position is thought to be important for directing the processing sites of the various precursor molecules to the active site of cathepsin E. Although the positions of Xaa and X`aa were occupied by various amino acids, including hydrophobic and aromatic amino acids, some of these had a negative effect, as typically observed when Glu/Arg and Pro were present at the P3 and P2 positions, respectively. Cathepsin D was much less active or was almost inactive in the processing of the precursors to neurotensin and related peptides as a result of the inability of the Pro-directed conformation of the precursor molecules to gain access to the active site of cathepsin D. Thus, the consensus sequence of precursors, Pro-Xaa-X`aa-hydrophobic amino acid, might not only generate the best conformation for cleavage by cathepsin E but might be responsible for the difference in specificities between cathepsins E and D.


INTRODUCTION

Many biologically active peptides are first synthesized as precursor molecules that must be processed correctly to yield the active peptides (reviewed in (1, 2, 3, 4) ). The bioactive peptide moiety in the precursor molecule often lies between paired dibasic motifs (Lys/Arg-Arg), occasionally between single basic residues, and rarely between hydrophobic recognition sites. Several endoproteolytic processing enzymes have been identified and characterized as being specified for these cleavage sites. Proteinases that are specific for the carboxyl side of pairs of basic amino acids include furin and prohormone convertases and they belong to the family of serine proteinases(2, 3, 4) . Precursors to some bioactive peptides are known to be processed by different types of proteinase. The involvement of aspartic proteinases has been demonstrated in some cases, for example the processing of proopiomelanocortin and the precursors to NT (^1)and related peptides. Although an aspartic proteinase, proopiomelanocortin-converting enzyme, has been isolated and characterized(5) , the proteinase(s) that processes the precursors to NT and related peptides has not been identified since pepsin has been used to mimic the processing proteinase(s) in most studies of the processing of these precursors(6, 7, 8, 9, 10, 11) .

To date, three types of intracellular aspartic proteinase, apart from proopiomelanocortin-converting enzyme, have been identified, namely cathepsin D, cathepsin E, and renin (reviewed in (12, 13, 14, 15, 16, 17) ). Cathepsins D and E have general proteolytic activities against protein substrates, such as hemoglobin and albumin, at acidic pH(12, 17) , while renin has a strict specificity for angiotensinogen at neutral pH (13) . Therefore, cathepsins D and E are thought to be able to process certain types of precursor including the NT/NMN precursor. Indeed, such processing activity was reported for cathepsin D, although the rate of hydrolysis was low(10) . Nonetheless, some important differences have been found between cathepsins D and E. While cathepsin D is a typical lysosomal enzyme, cathepsin E has been shown to be localized in the endoplasmic reticulum, in endosomes, or in other compartments where the processing of precursors is thought to occur(18, 19, 20, 21) . Moreover, as we have reported recently, cathepsin E has a unique structure among aspartic proteinases (22) and it is very active against certain types of peptide at weakly acidic pH, such as pH 5, with rates of hydrolysis several hundred-fold higher than those of reactions catalyzed by cathepsin D(23) . Since these results suggested that cathepsin E might be a more plausible candidate for a processing proteinase in some cases, we thought it appropriate to investigate the catalytic activity of cathepsin E with various precursor molecules.

In this study, we have purified cathepsin E from monkey intestine and examined its processing activity with several chemically synthesized peptides that included paired basic residues and other processing sites. Cathepsin D, renin, and pepsin were used for comparison. Cathepsin E processed precursors to NT and several other peptides much more rapidly and more specifically than did cathepsin D and other aspartic proteinases. An essential sequence in precursors that are processed by cathepsin E is proposed.


EXPERIMENTAL PROCEDURES

Materials

Biologically active peptides were purchased from Peptide Institute, Inc. (Minoh-shi, Japan), Peninsula Laboratories, Inc. (Belmont, CA), and Sigma. Fragments of the precursors to NT and other bioactive peptides were synthesized by Kurabo Co. (Osaka, Japan) and Bio-Synthesis, Inc. (Lewisville, TX). Porcine pepsin and renin were obtained from Sigma. Monkey cathepsin D was purified from spleen by the method of Tanji et al.(24) . The major isoform was used in the present study.

Purification of Monkey Cathepsin E

Cathepsin E was purified from the intestinal mucosa (duodenum and jejunum) of Japanese monkey (Macaca fuscata). The mucosa was homogenized in 0.01 M sodium phosphate buffer, pH 7.0, and the supernatant obtained by centrifugation at 15,000 g for 1 h was used for subsequent purification. The pH of the supernatant was lowered to 3.5 by the addition of formic acid, and the solution was then incubated at 37 °C for 1 h. The precipitate that formed was removed by centrifugation. Affinity chromatography on concanavalin A-Sepharose and then on pepstatin-Sepharose was carried out essentially as described by Tanji et al.(24) . Final purification of cathepsin E was achieved by fast protein liquid chromatography on a MonoQ column (HR 5/5,; Pharmacia LKB Biotech., Uppsala, Sweden) in 0.02 M Tris-HCl buffer, pH 8.0, with elution with a linear gradient of NaCl. Cathepsin E was eluted as a single peak between 0.3 and 0.4 M NaCl. The purified enzyme gave a single band of about 76 and 38 kDa by SDS-PAGE under non-deducing and reducing conditions, respectively, showing that the enzyme consists of two identical monomers (Fig.1). From 90 g of intestinal mucosa, we obtained 15 µg of cathepsin E with an overall yield of 20%. Purification was completed within 2 days, and the present procedure appears to be useful for studies of cathepsin E from primates. The amino acid composition of monkey cathepsin E closely resembled that of human cathepsin E (25) but differed from that of monkey cathepsin D(24) , especially in the levels of Lys, Leu, and Phe. The purified cathepsin E hydrolyzed various protein and peptide substrates similarly to cathepsin E from other animal sources(17) . The best peptide substrate was substance P, followed by eledoisin and neurokinin A (Fig.2).


Figure 1: SDS-PAGE of purified cathepsin E (E) and cathepsin D (D) from monkey. Proteins were subjected to electrophoresis under reducing (lanes1 and 2) and non-reducing (lanes3 and 4) conditions as described by Laemmli (51) and stained with Coomassie Brilliant Blue R-250. Since most cathepsin D exists in a two-chain form that consists of one heavy (30 kDa) and one light (15 kDa) chain, two bands were detected. In lane4, monkey pepsin A (35 kDa) (52) was added to the preparation of cathepsin E as a standard protein. The band of pepsin is shown by an arrow with a letterP.




Figure 2: The sequences and sites of cleavage by cathepsins E and D of various biologically active peptides. Residues at P1 and P1` positions are boxed. NH(2), shown in letters of reducedsize, indicates an amide group at the carboxyl terminus of a peptide. An arrow shows the cleavage sites. The rates of hydrolysis are given in nmol/min/µg protein at pH 5.0, except for the case of beta-endorphin, in which hydrolysis occurred maximally at pH 4.0; the values at pH 4.0 are given. The major cleavage site of beta-endorphin by cathepsin E was the Leu-Phe^18 bond, and the minor cleavage (at about 25% of the rate of the major cleavage) occurred at the Thr-Leu bond. The rate of hydrolysis given is that of the major cleavage. E, cathepsin E; D, cathepsin D; FGF, fibroblast growth factor.



Hydrolysis of Peptides

The procedure was based on that described in our previous reports(17, 23) . Details were as follows unless otherwise specified. The reaction mixture contained 0.2 M buffer at an appropriate pH, 50 µM peptide, and an appropriate amount of enzyme. The total volume was 20 µl. The reaction mixture was incubated at 37 °C for 1-6 h, and the reaction was stopped by the addition of 60 µl of 0.2 M Tris-HCl buffer, pH 8.0, or 3% perchloric acid. After removal of any precipitated material by centrifugation, each reaction mixture was subjected to high pressure liquid chromatography on a column (0.46 cm, inner diameter, 25 cm) of ODS-120T (Tosoh Corp., Tokyo) that had been equilibrated with 0.1% trifluoroacetic acid. The column was eluted with a linear gradient of acetonitrile that contained 0.1% trifluoroacetic acid. Each peptide was subjected to amino acid analysis and determination of the amino and/or carboxyl-terminal sequences by aminopeptidase M and/or carboxypeptidase Y to determine the sites of cleavage of substrate peptides.


RESULTS

Processing of the NT/NMN Precursor

Demonstrating a novel role for cathepsin E, we showed that the enzyme was capable of processing a group of precursors to bioactive peptides. First, we examined the processing of the NT/NMN precursor by cathepsin E since the involvement of a pepsin-like proteinase was previously anticipated in the processing of this precursor(10, 11) . NT and NMN are 13- and 6-residue peptides, respectively, which are located in the carboxyl-terminal region of a common precursor. There are Lys-Arg dibasic motifs on both sides of these two peptides (Fig.3, (26) and (27) ). Four peptides that included NT and/or NMN and a dibasic motif(s) were synthesized. Two synthetic oligopeptides, namely NT/NMN precursors 142-151 and 155-169, which contained one dibasic motif in their sequences, were cleaved very efficiently and specifically by cathepsin E at the bonds that connected an active peptide moiety and a basic residue, namely at the Leu-Lys and Leu-Lys bonds, respectively (Fig.3). The optimal pH was around 4.5-5. The rates of hydrolysis of these two precursor peptides by cathepsin E were very similar (Table1). However, when the Michaelis-Menten kinetics of two precursor peptides were compared, peptide 155-169 gave 15-100-fold lower K values at three different pH values than peptide 142-151, and the reverse relationship was found in the case of the K values (Table2). The similarity in the rates of hydrolysis of these two peptides can be explained by the comparable values of K/K. These precursor peptides were also hydrolyzed by porcine pepsin, although the rate of hydrolysis of peptide 155-169 was rather low. Cathepsin D and renin were scarcely able to hydrolyze any of these peptides. The NT/NMN precursor 134-147, which contained the third dibasic motif, Lys-Arg, was not cleaved by cathepsin E or by other aspartic proteinases. The cleavage by cathepsin E of a larger 24-residue peptide (NT/NMN precursor 142-165) was examined next. The sites of cleavage were the same as those of the two oligopeptides described above, but an additional cleavage occurred at the Leu-Tyr bond ( Fig.3and 4). Since smaller NTs, such as NT 6-13, have been found in intact cells(11) , cleavage at the Leu-Tyr bond might generate an intermediate between intact NT and NT 6-13. Such amino-terminal trimming of NT does not affect the biological activity of NT(11) . With peptide 142-165, the rates of hydrolysis at the two major cleavage sites, namely at the Leu-Lys and Leu-Lys bonds, were very different. The cleavage at the Leu-Lys bond preceded by far the cleavage at the Leu-Lys bond (Fig.4). The difference might be explained by the lower K value for cleavage of the Leu-Lys bond than that for cleavage of the Leu-Lys bond, as described above.


Figure 3: The carboxyl-terminal amino acid sequence of the precursor to rat NT/NMN and sites of cleavage by cathepsin E. The numbering is based on the results of Kislauskis et al.(27) . NT and NMN are boxed, and dibasic motifs on either side of the active moieties are shaded. The sequence of NT 6-13 is also shown. Four kinds of synthetic peptide and their sites of cleavage by cathepsin E are shown in the lower part of the Fig.








Figure 4: Time course of the hydrolysis of NT/NMN precursor peptides by cathepsin E. A dashedline indicates the change in the amount of the intact peptide 142-165. The sequence of the peptides and the cleavage sites are shown in Fig.3. , NT/NMN precursor peptide 142-162; bullet, peptide 142-147; , peptide 148-162; up triangle, filled, peptide 152-162.



Processing of Precursors to Xenopsin and Other Bioactive Peptides

Several bioactive peptides are known that are structurally related to NT and NMN (for example, xenopsin and angiotensin I)(11) . The processing sites of their precursors consist, however, of hydrophobic residues and differ from those of the NT/NMN precursor. Synthetic precursors to xenopsin and angiotensin I were exposed to cathepsin E and other aspartic proteinases (Table1). The precursor to xenopsin was efficiently processed by cathepsin E optimally at around pH 5, while other aspartic proteinases were almost inactive against this precursor. Porcine angiotensinogen was processed by cathepsin E more efficiently than the precursor to xenopsin, presumably due to the relatively high K value for its hydrolysis. However, as hitherto well known, this precursor was also processed by other aspartic proteinases such as renin(13, 28) . The difference between cathepsin E and renin was found in that cathepsin E was maximally active against angiotensinogen at around pH 5 and almost inactive at pH 7, while renin was much more active at neutral pH and less active at around pH 5.

From the results of hydrolysis of the precursors to NT and related peptides, it was clear that cathepsin E had a generally high ability to process these precursors in the weakly acidic pH region. Cathepsin E appeared to recognize the structures of NT and related peptides that are specified by the occurrence of Pro and a hydrophobic amino acid at the P4 and P1 positions, respectively (Fig.5). Such structural features were also found in substance P, the best peptide substrate for cathepsin E. Thus, the Pro-Xaa-X`aa-HBaa sequence seemed to be essential for cleavage by cathepsin E of precursors. We searched a protein data base and found some additional precursors that have the consensus sequence. One of these was proopiomelanocortin. In this precursor, the sequence that includes the processing site is Pro-Leu-Glu-Phe-Lys-Arg. This precursor was correctly processed by cathepsin E, a result that supports the present hypothesis (Table1). Cathepsin D and pepsin also processed this precursor. However, the cleavage of proopiomelanocortin 34-43 by cathepsin D and pepsin occurred at two sites, namely at the Phe-Lys and Glu-Phe bonds, while the cleavage by cathepsin E was restricted to the Phe-Lys bond. Since the Glu-Phe bond was not an appropriate processing site, the specificity of cathepsin D and pepsin was low as compared to that of cathepsin E. Processing of some other precursors with the consensus sequence, such as precursors to atrial natriuretic factor and vasoactive intestinal polypeptide, was not catalyzed by cathepsin E or by the other aspartic proteinases tested.


Figure 5: Structural characteristics of substance P, various precursors, and their mutants that were hydrolyzed by cathepsin E. Substance P, porcine angiotensinogen 1-14 (acetylated form), and human angiotensinogen 1-13 were commercial products. Other peptides were synthesized on the basis of the sequence data for the rat NT/NMN precursor(27) , canine xenopsin precursor(53) , and cattle proopiomelanocortin(54) . Residues at the P4 and P1 positions are boxed, and dibasic motifs are shaded. The sequences of the active peptide moieties are shown in normalcapitalletters, and those of the other parts are shown in italicletters. Residues in filledboxes are substituted residues. NH(2) at the carboxyl terminus of substance P represents an amide moiety. An arrow shows the cleavage sites. The rate of hydrolysis of each peptide by cathepsin E at pH 5 is given in nmol/min/µg protein. UC, uncleaved.



Effects on Processing of the Substitution of Amino Acids in the Consensus Sequences of Precursors

The effects of amino acid substitutions in the consensus sequences of precursor molecules were examined in the NT/NMN precursor 142-151 (Table1). Substitution of Leu at the P1 position by one of non-hydrophobic amino acids such as Ser completely hindered the hydrolysis of the precursor by cathepsin E and other aspartic proteinases. Substitution of Pro at the P4 position by Val and by Ser did not change the rate of hydrolysis by cathepsin E, but substitution by Glu decreased the rate of hydrolysis about 3-fold. These substitutions at the P4 position increased the rates of hydrolysis by cathepsin D. In particular, in the Pro Val mutant, rates of hydrolysis were 50-100-fold higher than in the absence of the substitution. Although, as shown in the previous section, the NT/NMN precursor 142-151 was hydrolyzed by cathepsin E about 500-fold (pH 5) and 70-fold (pH 4) more efficiently than by cathepsin D, and the rates of hydrolysis of the Pro Val mutant by cathepsins E and D were similar (pH 5) or identical (pH 4). Thus, the different effects of the replacement of Pro on cleavage by cathepsins E and D were clear. The native structure of the precursor, which we can call the Pro-directed structure, seems to interact efficiently with cathepsin E but not with cathepsin D. Substitutions at positions other than the P1 and P4 positions also affected the rate of hydrolysis. Substitutions at the P3 position by Glu and by Arg and at the P3` position by one of basic amino acids such as His and Lys reduced the rates of hydrolysis by cathepsin E and other aspartic proteinases. The negative effect of a Tyr Glu substitution at the P3 position was particular noteworthy. The increase in K was considerable in each case (Table2). No hydrolysis was observed when Ile at the P2 position was replaced by Pro.


DISCUSSION

Precursors to some bioactive peptides are known to be processed by aspartic proteinases(5, 6, 7, 8, 9, 10, 11) . However, pepsin has been used as a model processing proteinase in most relevant studies, and the actual proteinases have not been identified. In the present study, we examined the processing activity of cathepsin E in a comparison with the activities of other aspartic proteinases, such as cathepsin D. In our experiments, we used various synthetic peptides composed of 8-24 residues that were equivalent to parts of natural precursors. Although natural precursors are thought to be processed in more complicated systems, the experiments using synthetic oligopeptides should yield results that are useful for understanding the basic mechanism of the processing of native precursor molecules.

We showed for the first time that cathepsin E processes a group of precursors to bioactive peptides, such as NT, NMN, and xenopsin, with much higher activity and specificity than do cathepsin D and other aspartic proteinases. Since these precursors are thought to be processed on their way from the endoplasmic reticulum to secretory vesicles (1) and, occasionally, in acidified vesicles(29) , the maximal activity of cathepsin E at weakly acidic pH and its localization in the endoplasmic reticulum, endosomes, or other compartments (18, 19, 20, 21) favor its reaction with precursor molecules. Cathepsin E is also able to process angiotensinogen, as well as other aspartic proteinases such as renin(13, 28) , although their optimal pH values differ significantly. Cathepsin E might be partly involved in the intracellular processing of angiotensinogen in various cells and tissues(30) , although it may be inactive in processing angiotensinogen at neutral pH in the serum. Cathepsin E processes various precursors by recognizing the structural features of active peptide moieties, which are specified by the occurrence of Pro and a hydrophobic residue at the P4 and P1 positions, respectively. The consensus sequence is Pro-Xaa-X`aa-HBaa at the P4 through P1 positions (Fig.5). Let us consider the significance of this consensus sequence. First, a hydrophobic (or aromatic) residue at the P1 position seems to be essential. To date, the proteolytic specificity of cathepsin E has been investigated by several authors using various proteins and peptides, such as the B-chain of insulin (23, 31, 32, 33, 34) and many kinds of biologically active peptides(23) . The P1 and P1` positions of the cleavage sites in most of these cases have been occupied by hydrophobic amino acids. This specificity is common to aspartic proteinases such as cathepsin D (12) and pepsin (35, 36) and was the case in the present study for the hydrolysis of various bioactive peptides that included substance P and related tachykinins (Fig.2). The P1` residue, however, was shown not to be absolutely hydrophobic since precursors such as the NT/NMN precursor having a non-hydrophobic P1` residue were efficiently processed by cathepsin E.

The most interesting feature of the consensus sequence of the precursor molecules is the occurrence of Pro at the P4 position. Pro in a peptide or protein forms an imido bond and then restricts the conformation of a peptide or protein. The active site of cathepsin E is thought to be configured such that it can interact with the Pro-directed conformation of a substrate peptide. Pro at the P4 position has been shown to be favorable for the hydrolysis of synthetic chromogenic substrates for cathepsin E(37, 38) . The present study showed, however, that Pro at the P4 position has a positive effect but is not essential for peptide hydrolysis since other amino acids, such as Ser and Val, were shown to be able to replace Pro. This result seems reasonable since Ser or Val can maintain the conformation of a peptide such that it will fit into the active site as a consequence of the flexibility of the alpha peptide bond. Contrary to the case with cathepsin E, the Pro-directed conformation of a substrate peptide appeared to be very unfavorable for cleavage of a peptide by cathepsin D. Substitution of Pro by another amino acid allowed cathepsin D to hydrolyze the peptide. The more flexible alpha peptide bond of a substituted amino acid might change the conformation of a substrate peptide to allow it to fit into the active site of cathepsin D. Thus, Pro at the P4 position causes the distinct difference in the specificity for peptide-bond hydrolysis between cathepsins E and D, probably as a result of the inflexible imido bond.

In our present hypothesis, a substrate peptide for a cathepsin is expected to be long enough to introduce as many as nine residues into subsites S6 through S3` of the active site of the enzyme(23, 39) . In shorter synthetic chromogenic peptides that can serve as substrate for cathepsin D, the effect of Pro at the P4 position is unclear(40, 41) . Therefore, Pro at the P4 position in native precursors may play a definitive role in the processing of such precursors. The processing sites of native precursors have been shown to be located in or immediately adjacent to a beta turn(42) , as is also the case in the NT/NMN precursor(11) . Pro might well be responsible for such a characteristic structure since Pro is known to have the highest potential for formation of a beta turn(43) . Indeed, a high frequency of Pro residues around the processing sites of precursor molecules has been reported(44) . Therefore, in the native precursors preferred by cathepsin E, Pro at the P4 position may be primarily important for directing the conformation of the substrate to the active site of cathepsin E. In such large peptides, replacement of Pro by another amino acid might inhibit the processing of the precursor molecules.

Although the consensus sequence is thought to be essential for the processing by cathepsin E, there are a few exceptions. For example, precursors to atrial natriuretic factor and vasoactive intestinal polypeptide, which have sequences of Pro-Arg-Ser-Leu and Pro-Val-Pro-Val, respectively, are not processed by cathepsin E. This failure might be due to steric hindrance by Arg and Pro at the P3 and P2 positions, respectively, as shown by the significant decrease in the rate of hydrolysis of Arg and Pro mutants of the NT/NMN precursor. Arg and Pro at the P3 and P2 positions, respectively, are known to hinder the hydrolysis of peptides by pepsin(35) .

We cannot ignore the diversity of systems that are involved in the processing of precursors to bioactive peptides. There are, as is well known, multiple processing proteinases. To date, precursors with processing sites that consist of paired basic residues are known to be processed by a group of serine proteinases, such as furin and prohormone convertases(1, 2, 3, 4) . These proteinases recognize a paired basic motif and a preceding basic residue(s), for example, in the sequence Arg-Xaa-Lys/Arg-Arg(2, 4, 45, 46, 47) . Since the NT/NMN precursor contains a Lys-Arg motif but lacks Arg at the P4 position, furin and prohormone convertases may be inactive or less active against this precursor. In the case of a precursor that has only a dibasic motif, a furin-like proteinase has been reported to be inactive(48) . However, different proteinases may process an identical precursor molecule. As shown in the present report, proopiomelanocortin 34-43 was processed by cathepsin E, while the dibasic motif of this precursor is known to be processed by other enzymes, such as prohormone convertases(45, 47) , and proopiomelanocortin-converting enzyme(5) . The occurrence of different types of proteinase for processing of the same precursor might guarantee the complete processing of the precursor in a living cell. Indeed, the processing of precursors has been reported to proceed via different pathways depending on environmental conditions such as pH (49) .


FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-568-61-2891; Fax: 81-568-63-0085.

^1
The abbreviations used are: NT, neurotensin; HBaa, a hydrophobic amino acid; NMN, neuromedin N.


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