©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of a Vasoactive Intestinal Polypeptide-degrading Endoprotease from Porcine Antral Mucosal Membranes (*)

(Received for publication, November 18, 1994; and in revised form, January 13, 1995)

Gwang-Ho Jeohn Kenji Takahashi (§)

From the Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A neutral endoprotease was isolated from porcine antral mucosa and purified to homogeneity as examined by SDS-polyacrylamide gel electrophoresis (PAGE). Throughout the purification, t-butyloxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumaryl-7-amide (MCA) was used as a substrate, which was found to be hydrolyzed specifically by the enzyme at the Arg-Arg bond. Unexpectedly, however, the enzyme was also found to hydrolyze vasoacive intestinal polypeptide (VIP) fairly specifically and more efficiently when various neuropeptides and related peptides were examined as substrates. It could degrade VIP by cleaving three peptide bonds not containing an arginine residue(s) with K = 7.7 times 10M and k/K = 7.4 times 10^6M s (at pH 7.6 in the presence of 0.1% Lubrol PX), whereas only secretin, substance P, and a few others were hydrolyzed at much slower rates among the various peptides examined. Both activities toward the MCA substrate and VIP behaved in parallel throughout the purification procedures and showed essentially the same pH optimum and susceptibility toward various inhibitors and detergents. Therefore, both activities are thought to be due to the same enzyme. This endoprotease required 0.001% or a higher concentration of a detergent such as Lubrol PX or Triton X-100 for its maximal activity. Its optimum pH was about 7.5 and the molecular weight was estimated to be approximately 37,000 by SDS-PAGE. This enzyme was strongly inhibited by serine protease inhibitors such as diisopropylfluorophosphate and phenylmethanesulfonyl fluoride. It was also inhibited by p-chloromercuribenzoic acid, but not by some other cysteine protease inhibitors. Therefore, the enzyme appears to be most likely a kind of serine protease although its possibility as a cysteine protease cannot be completely excluded. Analysis of its cleavage specificity toward various oligopeptides indicated the possibility that the protease might recognize a specific amino acid sequence(s) and/or conformation in the vicinity of the cleavage site of the target peptide. Various characteristics of the endoprotease suggest that it is a novel membrane-bound neuropeptide-degrading endoprotease fairly specific for VIP.


INTRODUCTION

Vasoactive intestinal polypeptide (VIP) (^1)is a 28-amino-acid residue neuropeptide, which plays many physiological roles in the gut and nervous systems(1, 2, 3, 4) . VIP is found in all layers of the gut including stomach mucosa and membranes and is known to be a physiological mediator for relaxation of gastric smooth muscle and for pepsinogen release in stomach mucosa(4, 5, 6, 7, 8) . The physiological mechanism of inactivation of VIP has not been well clarified, but the primary pathway is thought to involve proteolytic degradation. Several peptidases which were thought to be related to inactivation of VIP have been studied including mast cell tryptase and chymase(9) , enkephalinase(10) , and gastric muscle membrane-associated peptidase (5) . However, there has been no report so far of a neuropeptide-degrading protease which can specifically degrade VIP.

The present study was initiated in an attempt to isolate and characterize such a protease(s) that might specifically degrade VIP and/or related neuropeptides. This type of protease is thought to be present in minute quantity in the tissue, and often in the membrane-bound form. Therefore, special care should be taken to minimize proteolytic degradation that is apt to occur during the isolation procedures from gastric mucosa which contains lysosomal and a variety of other proteases(11, 12, 13, 14, 15, 16) . Thus, we used density gradient fractionation in the initial stage of the isolation procedure to minimize proteolytic degradation of the target protease(s), especially by lysosomal proteases. Further, we first chose t-butyloxycarbonyl(Boc)-Arg-Val-Arg-Arg-4-methylcoumaryl-7-amide (MCA) as a routine substrate since VIP and related neuropeptides generally contain several basic residues, often including basic amino acid pairs, at which sites cleavages were expected to occur, and assay was done by HPLC analysis of the cleavage products of the synthetic peptide MCA which is resistant to the action of aminopeptidase unlike simple oligopeptides like VIP.

Thus, we could finally isolate a novel membrane-bound neutral endoprotease from porcine antral mucosa which can fairly specifically degrade VIP as well as Boc-Arg-Val-Arg-Arg-MCA, and here we report its enzymatic characteristics.


EXPERIMENTAL PROCEDURES

Materials

Fresh porcine stomachs were obtained from Shibaura Hormone Manufacturing Co. (Tokyo, Japan). Peptide MCA substrates, porcine VIP, porcine secretin, substance P, neurokinin A, [Arg^8] vasopressin, human endothelin 1, bovine adrenal medulla dodecapeptide (BAM-12P), alpha-neoendorphin, neurotensin, dynorphin A, L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane (E-64), leupeptin, chymostatin, bestatin, diprotin A, arphamenine A, and pepstatin A were purchased from the Peptide Institute Inc. A silver staining kit and iodoacetic acid from Wako Pure Chemical Ind. DEAE-cellulose (DE52) was obtained from Whatman, and p-chloromercuribenzoate (PCMB)-agarose and a BCA protein assay kit from Pierce. Sephacryl S-200 and a Mono-Q HR5/5 column were from Pharmacia Biotechnology Inc. Somatostatin 28, oxidized bovine insulin B chain, bovine pancreatic trypsin inhibitor (Kunitz), carbonic anhydrase, diisopropylfluorophosphate (DFP), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-tosyl-L-lysine chloromethyl ketone (TLCK), phenylmethanesulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), and o-phenanthroline were purchased from Sigma. Lubrol PX, Triton X-100, sodium cholate, EDTA, benzamidine hydrochloride, and 2-mercaptoethanol were from Nacalai Tesque Inc. Human progastrin peptide was synthesized in our laboratory using an Applied Biosystems peptide synthesizer model 431A and purified by HPLC. Aminopeptidase B was purified by successive chromatographies on DE52, Sepharose CL-6B, TSKgel butyl-Toyopearl 650, concanavalin A-Sepharose in our laboratory. A mixture of molecular weight marker proteins for SDS-polyacrylamide gel electrophoresis (PAGE) was from Bio-Rad. Other reagents used were of the highest grade available.

Subcellular Fractionation

All steps were performed at 4 °C unless otherwise specified. Porcine antral mucosa (25 g) was carefully scraped from fresh stomachs and homogenized in 250 ml of 10 mM Tris-Cl (pH 7.2) containing 0.27 M sucrose, 1 mM PMSF, and 10 µM leupeptin (TS-PL buffer) first in a Waring blender (15 s times 3 times) and then in a Teflon homogenizer. The homogenate was filtered through 4 layers of surgical gauze to remove cell debris and nuclei, and the filtrate was centrifuged at 500 times g for 10 min. The supernatant was centrifuged at 20,000 times g for 30 min. The pellet was suspended in TS-PL buffer and recentrifuged at 10,000 times g for 20 min. The fluffy material found on top of the compact vesicle pellet was gently swirled and removed, and the remaining pellet fraction was suspended in 10 ml of 0.27 M sucrose in 10 mM Tris-Cl (pH 7.2) and gently homogenized. This vesicle fraction was used in the subsequent sucrose density gradient centrifugation.

Sucrose Density Gradient Centrifugation

2.1 M sucrose solution was added to 4.5 ml of the above vesicle fraction to a final volume of 10 ml and a final sucrose concentration of 1.2 M in 10 mM Tris-Cl (pH 7.2). This was layered on a stepwise gradient composed of 0.27 M (6 ml), 0.8 M (6 ml), 1.45 M (4 ml), 1.7 M (4 ml) and 2.0 M (2 ml) sucrose in 10 mM Tris-Cl (pH 7.2). After centrifugation for 10 h at 100,000 times g in a Hitachi RPS 27-2 rotor, 1.5-ml fractions were collected from the bottom, and density was measured by a refractometer.

Enzymatic Activity Assay

All enzymatic reactions were done at 37 °C. The Boc-Arg-Val-Arg-Arg-MCA cleaving activity was assayed in a reaction mixture containing 0.1 mM substrate, 50 mM Tris-Cl (pH 7.6), 0.1% Lubrol PX, and 1 mM CaCl(2). The cleavage products were analyzed by an Applied Biosystems 130A separation system using an Aquapore OD-300 C(18) column (2.1 times 30 mm) eluted with a gradient of 0-45% acetonitrile in 0.1% trifluoroacetic acid. If necessary, the enzymatic reaction was done in 200 µl of the above reaction mixture containing 3 units of aminopeptidase B (1 unit of the activity was defined as the amount of the enzyme to liberate 1 µmol of AMC/min in the reaction mixture containing 0.1 mM Arg-MCA and 50 mM Tris-Cl (pH 7.2)). After addition of 1 ml of the stop solution containing 100 mM sodium chloroacetate, 20 mM sodium acetate, and 0.4%(v/v) acetic acid, the amount of AMC produced by the aminopeptidase reaction was measured fluorometrically with an excitation wavelength of 370 nm and an emission wavelength of 460 nm in a Hitachi fluorescence spectrophotometer 650-10S.

Aminopeptidase B activity was assayed by the method of Usui et al.(17) with modifications in 200 µl of a reaction mixture containing 0.1 mM Arg-MCA and 50 mM potassium phosphate (pH 6.5), and the amount of AMC produced was measured fluorometrically.

Acid phosphatase activity was assayed by the modified Lowry method (18) with 10 mMp-nitrophenyl phosphate in 500 µl of 50 mM sodium acetate buffer (pH 5.4) at 37 °C for 1 h. After the reaction, 500 µl of 0.2 N NaOH was added, and the absorbance at 410 nm was measured.

Trypsin-like protease activity toward Boc-Gln-Gly-Arg-MCA was assayed fluorometrically as described (11) with 0.1 mM substrate in 200 µl of 50 mM Tris-Cl (pH 8.2).

Cysteine protease activity toward Boc-Leu-Arg-Arg-MCA was assayed with 0.1 mM substrate in 200 µl of 50 mM potassium phosphate (pH 6.5) and 1 mM 2-mercaptoethanol, and the amount of AMC produced was measured fluorometrically.

Protein Determination

Protein was determined by measuring the absorbance at 280 nm of the sample solution or by the method of Smith et al.(19) using the BCA reagent. The protein finally isolated was microquantitated on SDS-PAGE after silver staining by comparing with pancreatic trypsin inhibitor as a standard protein.

Purification of a VIP-degrading Enzyme

All procedures were performed at 4 °C. Ten volumes of 40 mM Tris-Cl (pH 7.6) containing 10 µM leupeptin and 1% Lubrol PX were added to the 1.5 ml of the active fraction obtained by sucrose density gradient centrifugation, and the enzyme was solubilized by magnetic stirring overnight.

The solubilized sample was centrifuged at 20,000 times g for 25 min in a Beckman 50.2 Ti rotor. The resulting clear supernatant was dialyzed against 40 mM Tris-Cl buffer (pH 7.6), 0.2% Lubrol PX. The dialyzed sample was applied to a DE52 column (1.5 times 10 cm) equilibrated with 40 mM Tris-Cl buffer (pH 7.6), 0.2% Lubrol PX, and eluted with a linear gradient of 0-0.5 M NaCl. The active fractions were pooled and concentrated to 1 ml, and applied to a Sephacryl S-200 column (1 times 145 cm) equilibrated and eluted with 40 mM Tris-Cl buffer (pH 7.6) containing 0.2 M NaCl and 0.05% Lubrol PX.

The pooled active fraction from the Sephacryl S-200 was applied to a Mono-Q/FPLC HR5/5 column and eluted with a linear gradient of 0-0.5 M NaCl in 50 mM Tris-Cl (pH 7.6), 0.02% Lubrol PX. The active fractions were pooled and applied to a PCMB-agarose affinity column (0.9 times 3 cm) and eluted first with 50 mM 2-mercaptoethanol, 50 mM Tris-Cl (pH 7.6), 0.2 M NaCl, and then with 100 mM 2-mercaptoethanol, 50 mM Tris-Cl (pH 7.6), and 1.0 M NaCl. The active fractions were pooled and dialyzed against 40 mM Tris-Cl (pH 7.6).

Digestion of Oligopeptides and Analysis of the Cleavage Sites

Each peptide (1,000 pmol) was incubated in 20 µl of a reaction mixture containing 50 mM Tris-Cl (pH 7.6), 1 mM CaCl(2), 0.1% Lubrol PX, and 5.4 fmol of the purified enzyme. The peptide fragments produced were separated by HPLC with a Hitachi 655A-11/LC5000 system using a TSKgel ODS-120T reverse-phase column (0.46 times 25 cm). Elution was performed with a gradient from 0% acetonitrile in 0.1% trifluoroacetic acid to 50% acetonitrile in 0.08% trifluoroacetic acid and monitored at 215 and 280 nm. The amino acid composition of each peak was analyzed in an Applied Biosystems 420A derivatizer/analyzer.


RESULTS

Screening of a Novel Protease by Sucrose Density Gradient Centrifugation

In sucrose density gradient centrifugation (Fig. 1), the activity of acid phosphatase, a lysosomal marker enzyme, was mainly distributed in fractions 11-15 while the aminopeptidase activity toward Arg-MCA was mainly in fractions 12-16 and in 5-8 (Fig. 1B). Trypsin-like protease activity toward Boc-Gln-Gly-Arg-MCA from the vesicular source was rather low and was observed in a broad region (Fig. 1C), whereas much higher activity was observed in the microsomal fraction (distributed at density 1.23 g/ml) (data not shown). Its activity distribution was different from those of acid phosphatase and aminopeptidase B. Cysteine protease activity toward Boc-Leu-Arg-Arg-MCA, requiring a reducing agent, was also broadly distributed in fractions from 7 to 13 (Fig. 1C).


Figure 1: Isolation of a new protease activity from porcine antral mucosal vesicle fraction through sucrose density gradient centrifugation. A, sucrose density(circle) and protein(bullet). Sucrose density gradient centrifugation was done as described under ``Experimental Procedures.'' B, activities of aminopeptidase B(circle) and acid phosphatase (bullet). C, trypsin-like protease activity toward Boc-Gln-Gly-Arg-MCA(circle) and thiol protease activity toward Boc-Leu-Arg-Arg-MCA (bullet). D, activity of a novel protease. A novel endoprotease activity was screened with 1 mM Boc-Arg-Val-Arg-Arg-MCA as a substrate in 50 mM Tris-Cl (pH 7.2) containing 1 mM CaCl(2), 0.02% Lubrol PX, and 20 µM leupeptin with aminopeptidase B and the reaction products were analyzed fluorometrically as described under ``Experimental Procedures.''



To detect a novel protease, we used the synthetic peptide Boc-Arg-Val-Arg-Arg-MCA which has often been used as a substrate for furin and related proteases(20) . In addition to the fluorometric determination of AMC produced directly by enzymatic reaction, we analyzed the reaction products by HPLC or determined AMC after additional reaction with aminopeptidase B because some proteases may cleave at other site(s) than the Arg-MCA bond in the substrate(21) . Through this screening method we found a new proteolytic activity unexpectedly cleaving the Arg-Arg bond, but not the Arg-MCA bond, of Boc-Arg-Val-Arg-Arg-MCA. This activity was found in fraction 7 (at density 1.17-1.18 g/ml) (Fig. 1D), was not inhibited by leupeptin, and was clearly different from the activities of the trypsin-like protease(s), cysteine protease(s), and aminopeptidase(s) which were also found in gastric mucosal vesicle fractions.

Purification of the Novel Protease

When the novel protease fraction obtained by sucrose density gradient centrifugation was chromatographed on DE52, the activity cleaving the Arg-Arg bond of Boc-Arg-Val-Arg-Arg-MCA was obtained as shown in Fig. 2A. The active fractions (fractions 20-26) were pooled for the subsequent purification on Sephacryl S-200, which gave a single peak of activity (Fig. 2B). The pooled S-200 fraction (fractions 42-48) was purified further by Mono-Q/FPLC. Upon Mono-Q/FPLC, the enzyme was eluted at 220 mM NaCl as a single peak (Fig. 2C). Based on the preliminary analysis showing that this protease activity was strongly inhibited by PCMB, we then tried to purify the protease further by PCMB-agarose affinity chromatography. Many enzymes are eluted with 10-50 mM 2-mercaptoethanol from a PCMB-agarose or PCMB-Sepharose column (22, 23, 24) . Unusually, the present enzyme activity was not eluted with 50 mM 2-mercaptoethanol, but could be eluted with 100 mM 2-mercaptoethanol plus 1.0 M NaCl as shown in Fig. 2D.


Figure 2: Purification of a VIP-degrading endoprotease. A, DE52 chromatography (fraction size, 3 ml; flow rate, 0.5 ml/min). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer as described under ``Experimental Procedures.'' Fractions 20-26 were pooled. B, Sephacryl S-200 chromatography (fraction size, 2 ml; flow rate, 6 ml/h). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer. Fractions 42-48 were pooled. C, Mono-Q/FPLC (fraction size, 0.5 ml; flow rate, 0.5 ml/min). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer. Fractions 24-27 were pooled. D, PCMB-agarose affinity chromatography. The sample was loaded and washed in 50 mM of Tris-Cl (pH 7.6), 0.2 M NaCl, 0.02% Lubrol PX (a), and eluted first with 50 mM 2-mercaptoethanol, 50 mM of Tris-Cl (pH 7.6), 0.2 M NaCl (b), and then eluted with 100 mM 2-mercaptoethanol, 50 mM of Tris-Cl (pH 7.6), 1.0 M NaCl (c). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer. Fractions 22-24 were pooled and dialyzed against 40 mM Tris-Cl (pH 7.6).



Thus we finally isolated 0.4 µg of the purified enzyme from 21.6 mg of protein of the active fraction obtained by sucrose density gradient centrifugation of the antral vesicle fraction (Table 1). Its molecular weight was estimated to be approximately 37,000 by 12.5% SDS-PAGE under both reducing and nonreducing conditions (Fig. 3), indicating that it is composed of a single chain polypeptide. The protease activity coincided well with the band of the purified protein on 12.5% native-PAGE (Fig. 3).




Figure 3: PAGE of the purified endoprotease. Slab gel electrophoresis was performed by the method of Laemmli(35) , and the gel was stained using a silver staining kit. A, 12.5% SDS-PAGE of the endoprotease. B, 12.5% native-PAGE of the endoprotease. a, carbonic anhydrase; b, the endoprotease. C, analysis of the enzyme activity in the native-PAGE. The gel was sliced into 24 pieces and extracted overnight in 40 µl each of the assay buffer. The enzymatic activities toward Boc-Arg-Val-Arg-Arg-MCA (circle) and VIP (bullet) were assayed as described under ``Experimental Procedures.'' BPB, bromphenol blue.



Properties of the Novel Protease and Its Characterization as a VIP-degrading Protease

When various synthetic peptide MCAs were used as substrates, the purified protease showed distinct specificity as shown in Table 2. It cleaved on the N-terminal side of an arginine residue immediately preceding the MCA moiety in some of the MCA substrates (Table 2). The best substrate among them was Boc-Arg-Val-Arg-Arg-MCA and the enzyme cleaved the Arg-Arg bond, but the other sites were not cleaved at all.



On the other hand, when various oligopeptides including gastrointestinal peptides were examined as substrates, the purified protease cleaved only VIP most rapidly, and secretin at a slower rate which has structural similarities to VIP (25) and no other peptides were hydrolyzed significantly except that substance P and oxidized insulin B chain were hydrolyzed slowly (Table 3). Interestingly, VIP was mainly cleaved by this protease at three sites which had not been expected from its specificity toward the synthetic peptide MCA substrates as shown in Fig. 4, and no cleavage occurred in the other part of VIP including the Thr-Arg-Leu-Arg-Lys sequence of which the Arg-Lys and Thr-Arg bonds were expected to be cleaved by the enzyme as judged from its specificity toward MCA substrates. The cleaving activity of VIP at the three sites (Fig. 4) paralleled the Boc-Arg-Val-Arg-Arg-MCA cleaving activity throughout the purification by successive column chromatography on Sephacryl S-200, Mono-Q/FPLC, and PCMB-agarose.




Figure 4: HPLC profiles of a digestion mixture of VIP and the cleavage sites. 1,000 pmol of VIP was digested with 5.4 fmol of the purified enzyme for 1 h and the peptide fragments were analyzed as described under ``Experimental Procedures.'' A. HPLC pattern. B. Cleavage sites and the peptide fragments produced. The amino acid sequence of VIP is shown in one-letter amino acid code. The values in parenthesis indicate the extents of cleavage.



Kinetic parameters with VIP and Boc-Arg-Val-Arg-Arg-MCA as substrates were analyzed as shown in Table 4. These results indicated that the enzyme has much higher affinity and catalytic efficiency toward VIP than toward Boc-Arg-Val-Arg-Arg-MCA.



The protease activity toward Boc-Arg-Val-Arg-Arg-MCA was increased about eight times by the addition of Lubrol PX or Triton X-100, whereas sodium cholate was not effective and SDS showed inhibition at above 0.001% (Fig. 5A). Likewise, the protease activity toward VIP was dependent on Lubrol PX (Fig. 5A). This indicated that membrane components are necessary for the endoprotease to have maximal enzymatic activity and that the enzyme is indeed a membrane-bound protease. This protease activity toward VIP and Boc-Arg-Val-Arg-Arg-MCA showed an optimum at pH 7.5 (Fig. 5B).


Figure 5: Effects of various detergents and pH values on the VIP-degrading endoprotease activity. A, effects of various detergents. The activity was assayed as described under ``Experimental Procedures'' except for the addition of detergents: toward VIP with Lubrol PX (bullet) and toward Boc-Arg-Val-Arg-Arg-MCA with Lubrol PX (circle), Triton X-100 (up triangle), sodium cholate (), and SDS (box). B, effects of pH. The activity was assayed as described under ``Experimental Procedures'' except for the buffer used: toward VIP (bullet) and toward Boc-Arg-Val-Arg-Arg-MCA (circle). The pH was varied using the universal buffer(36) .



This protease was inhibited by serine protease inhibitors such as DFP and PMSF. The activity was completely inhibited by 10 mM DFP, although it was not so effective at 1 mM concentration. Interestingly, this protease was also inhibited by PCMB, chymostatin, and iodoacetic acid, but not by E-64 and leupeptin which are also cysteine protease inhibitors (Table 5). The activity was increased about 40% by 1 mM Ca ion, whereas it was strongly inhibited by metal ions such as Fe, Cu, Zn, and Hg.




DISCUSSION

Rapid and specific inactivation of a neuropeptide after its function is important and is thought to depend on the action of its inactivating enzyme(s). There have been many reports of different peptidases which may be involved in neuropeptide degradation(16, 26, 27, 28, 29, 30, 31, 32) , but there is not much convincing evidence as yet for any of them that it is specific for a single peptide. In the present study, we have attempted to isolate a novel protease(s) from porcine antral mucosa which may be involved in neuropeptide processing or degradation using a synthetic peptide MCA substrate, Boc-Arg-Val-Arg-Arg-MCA, and could finally isolate a 37,000 dalton neutral endoprotease, which was found to cleave VIP highly efficiently as well as specifically. This new endoprotease activity was distributed in a narrow range in the sucrose density gradient centrifugation as compared with other protease activities (Fig. 1), and showed the optimum activity at pH 7.5 (Fig. 5B), which is higher than that of secretory granules or lysosomes (pH 5.5-6.5). These results indicated the possibility that this enzyme might be associated with some specific membranous structures other than secretory granules or lysosomes. The activity was dependent on some detergents (Fig. 5A). This protease required 0.001% or a higher concentration of a detergent for maximal activity. The enzyme could be kept very stable when Lubrol PX was added to the enzyme solution, but it was very unstable without Lubrol PX (data not shown). Therefore the presence of certain membrane components in vivo seems to be very important for its activity and stability. These results also suggest strongly that the enzyme is bound to a specific membranous structure by hydrophobic interaction.

The endoprotease was inhibited by serine protease inhibitors; it was completely inhibited by 10 mM DFP or 1 mM PMSF. It was also inhibited strongly by PCMB and iodoacetic acid and had a high affinity toward a PCMB-agarose affinity column, whereas it was not inhibited by other cysteine protease inhibitors such as E-64 and leupeptin, and it did not require any reducing agent for its activity. Therefore, the present protease appears to be most likely a serine protease although its action as a cysteine protease cannot be completely excluded. This enzyme is thought to have an SH group(s) in the vicinity of the active site, the modification of which results in an extensive inactivation of the enzyme. The present protease showed a distinct substrate specificity as shown in Table 2and Table 3. The protease cleaved Boc-Arg-Val-Arg-Arg-MCA at the Arg-Arg bond. This cleavage pattern is distinct from that of furin and related proteases (20, 33, 34) . The same type of cleavage was also observed at the Lys-Arg and Thr-Arg bonds in Pyr-Arg-Thr-Lys-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA, respectively, but susceptibility was very low when compared with that of Boc-Arg-Val-Arg-Arg-MCA. On the other hand, when various peptides were used as substrates, VIP was found to be cleaved much more specifically than synthetic peptide MCA substrates (Table 4). Unexpectedly, VIP was cleaved at three sites (A-C sites in Fig. 4) not involving arginine residues. These results suggested the possibility that the Boc-Arg-Val-Arg-Arg-MCA cleaving activity, and the VIP-degrading activity might be due to different enzymes. However, each cleaving activity in VIP and the Boc-Arg-Val-Arg-Arg-MCA cleaving activity coincided well with each other in pH dependence (Fig. 5B) and the effects of inhibitors (Table 5). The protease activity toward VIP was strongly dependent on Lubrol PX as in the case of Boc-Arg-Val-Arg-Arg-MCA (Fig. 5A). Furthermore, upon native PAGE of the purified enzyme, both Boc-Arg-Val-Arg-Arg-MCA cleaving activity and VIP-degrading activity coincided practically completely with each other (Fig. 3). These results indicate that these activities are due to a single enzyme.

The present protease showed high substrate specificity, hydrolyzing only a few substrates. There appears to be some tendency that certain amino acid residues with bulky, but not aromatic, side chains, such as Gln, Met, Lys, Leu, etc., are preferred at the P(1) and often at the P(1)` positions. However, the amino acid residues at the cleavage sites and in their vicinities are fairly variable, and it is difficult to define clearly the subsite specificity of the enzyme. This enzyme might recognize a specific amino acid sequence(s) and/or conformation in the vicinity of the cleavage site of the target peptide. Taken together, the present enzyme is strongly suggested to be involved in the enzymatic inactivation of certain neuropeptides, especially VIP. However, further studies are necessary to obtain a definite conclusion on the physiological role and the substrate specificity of this novel proteinase.


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: Dept. of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Tel.: +81-3-5689-5607; Fax: +81-3-5802-2041.

(^1)
The abbreviations used are: VIP, vasoactive intestinal polypeptide; Boc, t-butyloxycarbonyl; MCA, 4-methylcoumaryl-7-amide; BAM-12P, bovine adrenal medulla dodecapeptide; E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane; PCMB, p-chloromercurybenzoic acid; BCA, bicinconinic acid; DFP, diisopropylfluorophosphate; TPCK, N--tosyl-L-phenylalanine chloromethyl ketone; TLCK, N--tosyl-L-lysine chloromethyl ketone; PMSF, phenylmethanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-4-methylcoumarin: FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Hideshi Inoue, Dr. Masashi Matsushima, and Yuichi Tsuchiya for valuable discussions on this work, and Yasuko Sakurai for amino acid analysis.


REFERENCES

  1. Schubert, M. L., and Makhlouf, G. M. (1993) Gastroenterology 104, 834-839 [Medline] [Order article via Infotrieve]
  2. Wang, Y., and Colon, J. M. (1993) Peptides 14, 573-579 [CrossRef][Medline] [Order article via Infotrieve]
  3. Said, S. I., and Mutt, V. (1970) Science 169, 1217-1218 [Medline] [Order article via Infotrieve]
  4. Keast, J. R., Furness, J. B., and Costa, M. (1985) J. Comp. Neurol. 236, 403-412 [Medline] [Order article via Infotrieve]
  5. Kobayashi, R., Chen, Y., Lee, T. D., Davis, M. T., Ito, O., and Walsh, J. H. (1994) Peptides 15, 323-332 [Medline] [Order article via Infotrieve]
  6. Ozaki, H., Blondfield, D. P., Mori, M., Sanders, K. M., and Publicover, N. G. (1992) J. Physiol. 447, 351-372 [Abstract]
  7. Tazi-Saad, K., Chariot, J., and Rozé, C. (1992) Peptides 13, 233-239 [Medline] [Order article via Infotrieve]
  8. Felley, C. P., Qian, J.-M., Mantey, S., Pradhan, T., and Jensen, R. T. (1992) Am. J. Physiol. 263, G901-907
  9. Caughey, G. H., Leidig, F., Viro, N. F., and Nadel, J. A. (1988) J. Pharmacol. Exp. Ther. 244, 133-137 [Abstract]
  10. Goetzl, E. J., Sreedharan, S. P., Turck, C. W., Bridenbaugh, R., and Malfroy, B. (1989) Biochem. Biophys. Res. Commun. 158, 850-854 [Medline] [Order article via Infotrieve]
  11. Uchino, T., Sakurai, Y., Nishigai, M., Takahashi, T., Arakawa, H., Ikai, A., and Takahashi, K. (1993) J. Biol. Chem. 268, 527-533 [Abstract/Free Full Text]
  12. Fruton, J. S. (1971) Enzymes 3, 119-164
  13. Koshikawa, N., Yasumitsu, H., Umeda, M., and Miyazaki, K. (1992) Cancer Res. 52, 5046-5053 [Abstract]
  14. Kageyama, T., and Takahashi, K. (1980) J. Biochem. (Tokyo) 87, 725-735 [Abstract]
  15. Woolley, D. E., Tucker, J. S., Green, G., and Evanson, J. M. (1976) Biochem. J. 153, 119-126 [Medline] [Order article via Infotrieve]
  16. Bunnett, N. W., Goldstein, S. M., and Nakazato, P. (1992) Gastroenterology 102, 76-87 [Medline] [Order article via Infotrieve]
  17. Usui, Y., Ohtomo, T., and Yoshida, K. (1982) Biochim. Biophys. Acta 719, 539-543
  18. Lowry, O. H. (1957) Methods Enzymol. 4, 371-372
  19. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, E. K., Fujimoto, E. K., Goeke, N. M., Olsen, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85 [Medline] [Order article via Infotrieve]
  20. Molley, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402 [Abstract/Free Full Text]
  21. Azaryan, A. V., and Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202 [CrossRef][Medline] [Order article via Infotrieve]
  22. Hirao, T., Hara, K., and Takahashi, K. (1984) J. Biochem. (Tokyo) 95, 871-879 [Abstract]
  23. Hosoi, K., Kobayashi, S., and Ueha, T. (1978) Biochem. Biophys. Res. Commun. 85, 558-563 [Medline] [Order article via Infotrieve]
  24. Tsuru, D., Fujiwara, K., and Kado, K. (1978) J. Biochem. (Tokyo) 84, 467-476 [Abstract]
  25. Taylor, I. L., and Mannon, P. (1991) Textbook of Gastroenterology (Yamada, T., Alpers, D. H., Owyang, C., Powell, D. W., and Silverstein, F. E., eds) Vol. I, pp. 24-49, J. B. Lippincott Co., Philadelphia
  26. Matsas, R., Fulcher, I. S., Kenny, A. J., and Turner, A. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3111-3115 [Abstract]
  27. Bunnet, N. W., Walsh, J. H., and Debas, H. T. (1990) Am. J. Physiol. 258, G143-151
  28. Terashima, H., Okamoto, A., Menozzi, D., Goetzl, E. J., and Bunnett, N. W. (1992) Peptides 13, 741-748 [Medline] [Order article via Infotrieve]
  29. Checler, F., Vincent, J. P., and Kitabgi, P. (1985) J. Neurochem. 45, 1509-1513 [Medline] [Order article via Infotrieve]
  30. Joudiou, C., Carvalho, K. M., Camarao, G., Boussetta, H., and Cohen, P. (1993) Biochemistry 32, 5969-5966
  31. Carvalho, K. M., Joudiou, C., Boussetta, H., Leseney, A.-M., and Cohen, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 84-88 [Abstract]
  32. Millican, P. E., Kenny, A. J., and Turner, A. J. (1991) Biochem. J. 276, 583-591 [Medline] [Order article via Infotrieve]
  33. Steiner, D. F., Smeekens, S. P., Ohagi, S., and Chan, S. J. (1992) J. Biol. Chem. 267, 23435-23438 [Free Full Text]
  34. Barr, P. J. (1991) Cell 66, 1-3 [Medline] [Order article via Infotrieve]
  35. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  36. Perrin, D. D., and Dempsey, B. (1974) Buffers for pH and Metal Ion Control, p. 48, Chapman & Hall Ltd., London

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.