©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of Gastric Trypsin from the Microsomal Fraction of Porcine Gastric Antral Mucosa (*)

Gwang-Ho Jeohn (1)(§), Shou Serizawa (1), Akihiro Iwamatsu (2), Kenji Takahashi (1)(¶)

From the (1)Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan and the (2)Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., Yokohama-shi, Kanagawa 236, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A gastric serine protease(s) was found in porcine gastric antral mucosa and was shown to be distributed in the endoplasmic reticulum (ER)-microsome fraction and also in the vesicle fraction. Two forms of the protease were purified over 6,000-fold from the ER-microsome fraction. Analyses of various molecular and enzymatic characteristics including the N-terminal and partial internal amino acid sequences of both forms revealed that they share the same properties and are indistinguishable from porcine pancreatic trypsin. This is the first time that trypsin or a protease almost identical with trypsin has been found to be present intracellularly in normal tissues.

The gastric trypsin activities from the ER-microsome and the vesicle fractions were located in distinct density regions upon density gradient centrifugation, which indicates association of the protease with different organelle membranes. Taken together, these results suggest that there may be a novel function of trypsin in the gastric mucosa; it might function as a specific degrading or processing enzyme as an intracellular protease.


INTRODUCTION

The gastric mucosa is thought to contain several endopeptidases involved in proteolytic processing or degradation of certain biologically important proteins and peptides and their precursors, since various bioactive peptides, including gastrin, somatostatin, vasoactive intestinal polypeptide (VIP),()and others, are known to be present in the stomach(1, 2, 3) . Previously, we found a serine protease trapped in -macroglobulin in the cytosol fraction of porcine gastric mucosa and suggested the possibility of its participation in the processing or degradation of certain bioactive peptides(4) . However, due to its apparent instability after release from -macroglobulin, no extensive characterization has been made so far. We also suggested similar roles for gastric cathepsin E at neutral pH(5) . There is, to our knowledge, however, no other report on the isolation and characterization of such endopeptidases from gastric mucosa except for a VIP-degrading endoprotease from antral mucosal membranes (6) that might be involved in the processing or specific degradation of biologically active proteins and peptides. On the other hand, it was reported that pancreatic trypsin inhibitor is present in mucus-producing foveolar cells in the stomach(7, 8, 9) . The presence of protease inhibitors such as pancreatic trypsin inhibitor and -macroglobulin suggests that they may play a role in regulating the activities of certain proteases, especially trypsin-like proteases, in the mucosa.

In the present study, we attempted to explore, isolate, and characterize such a trypsin-like protease(s) which may be related to proteolytic processing or degradation in vesicles or endoplasmic reticulum (ER) of gastric mucosa. We investigated the distribution of trypsin-like protease activity and could finally isolate and characterize a serine protease from the ER-microsome fraction of the gastric antral mucosa. In so far as it has been studied, the enzyme is indistinguishable from pancreatic trypsin, and we tentatively named it ``gastric trypsin.'' This is the first time that trypsin (or an enzyme almost identical with trypsin) has been found to exist intracellularly.


EXPERIMENTAL PROCEDURES

Materials

Fresh porcine stomachs were obtained from Shibaura Hormone Manufacturing Co. (Tokyo, Japan). Peptide 4-methylcoumaryl-7-amide (MCA) substrates, porcine VIP, bovine adrenal medulla dodecapeptide (BAM-12P), -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., and iodoacetic acid, a silver staining kit and Acromobacter protease I (lysyl endopeptidase) from Wako Pure Chemical Inc. DEAE-cellulose (DE52) was obtained from Whatman, and TSKgel butyl-Toyopearl 650 from Toso Corp. Sepharose CL-6B and benzamidine-Sepharose 6B were from Pharmacia Fine Chemicals, and a BCA protein assay kit from Pierce. NAP-5 column was from Pharmacia Biotechnology Inc. Porcine pancreatic trypsin, diisopropyl fluorophosphate (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 deoxycholate, EDTA, and benzamidine hydrochloride were from Nacalai Tesque Inc. [1,3-H]DFP (111 GBq/mmol) was purchased from DuPont NEN. Human progastrin peptide was synthesized in our laboratory using an Applied Biosystems peptide synthesizer model 431A and purified by HPLC. A mixture of molecular weight marker proteins for sodium dodecyl sulfate (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. Fresh porcine stomachs were washed well with chilled 10 mM Tris-Cl (pH 7.2) containing 0.27 M sucrose, and the antral mucosa (77 g) was carefully scraped and homogenized in 5 volumes 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 three times) and then in a Teflon homogenizer. The homogenate was filtered through four layers of surgical gauze to remove cell debris and nuclei and the filtrate was centrifuged at 500 g for 10 min. The supernatant was centrifuged at 20,000 g for 20 min. The pellet was suspended in TS-PL buffer and recentrifuged at 10,000 g for 20 min. The pellet was suspended in a 0.2 volume of 10 mM Tris-Cl (pH 7.2) containing 0.27 M sucrose. This vesicle fraction was used in the subsequent density gradient centrifugation. The supernatant at 20,000 g for 20 min was centrifuged for 60 min at 100,000 g, and the pellet was suspended into the same buffer and recentrifuged for 60 min at 100,000 g. This microsomal pellet was suspended in 0.2 volume of 10 mM Tris-Cl (pH 7.2) containing 0.27 M sucrose and also used in the subsequent density gradient centrifugation.

Sucrose Density Gradient Centrifugation

2.1 M sucrose solution was added to 4.5 ml of the vesicle or the microsome 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 36 h at 100,000 g (Hitachi RPS 27-2 rotor), 1.5-ml fractions were collected from the bottom.

Metrizamide-Sucrose Density Gradient Centrifugation

The sample was prepared according to the subcellular fractionation procedure described above except that Tris-Cl (pH 8.8) and 1 mM EDTA were used. The metrizamide stepwise gradient was composed of 0.10 M (5 ml), 0.15 M (5 ml), 0.23 M (5 ml), 0.3 M (8 ml), and 0.4 M (5 ml) metrizamide in 10 mM Tris-Cl (pH 7.2) and 1 mM EDTA, and the final osmolarity was adjusted with sucrose solution to 300 mosm/kg(10) . 10 ml of the vesicle fraction was layered on the metrizamide-sucrose stepwise gradient. After centrifugation for 1.5 h at 100,000 g (Hitachi RPS 27-2 rotor), 1.5-ml fractions were collected from the bottom. [H]DFP Labeling of Pancreatic Trypsin-50 µg of pancreatic trypsin was incubated for 2 h at 37 °C in 100 µl of a reaction mixture containing 40 mM Tris-Cl (pH 8.2), 1 mM CaCl, and 1 MBq of [1,3-H]DFP. After addition of 4.2 µl of 5 M NaCl, the solution was applied to a NAP-5 (Sephadex G-25) column (0.9 2.8 cm) equilibrated with 40 mM Tris-Cl (pH 8.2), 0.2 M NaCl, and 1 mM CaCl. Radioactivity of each fraction was measured in a liquid scintillation counter (Aloka model LSC700), and the radioactive fractions containing [H]DFP-labeled pancreatic trypsin were also analyzed by 12.5% SDS-PAGE and autoradiography. The [H]DFP-labeled pancreatic trypsin was used as an exogenous authentic enzyme. Subcellular Fractionation of the Antral Mucosa Supplemented with Exogenous [H]DFP-labeled Pancreatic Trypsin-[H]DFP-labeled pancreatic trypsin (corresponding to the radioactivity of 0.2 MBq) was supplemented additionally to 5 g of fresh antral mucosa, and the mucosa was homogenized in 5 volumes of TS-PL buffer. The subcellular fractionation was done as described above under the ``Subcellular Fractionation'' section except that each pellet of vesicle or microsome fraction was not washed. Radioactivity of the vesicle, microsome, and cytosol fractions was analyzed. Density Gradient Centrifugation of the Cellular Fractions Containing Exogenous [H]DFP-labeled Pancreatic Trypsin-It was analyzed using metrizamide-sucrose or sucrose density gradient fractionation to investigate whether the exogenous [H]DFP-labeled pancreatic trypsin can associate with vesicular and ER-microsomal membrane components. Sucrose or metrizamide-sucrose density gradient fractionation of the vesicle or microsome fraction was done as described above under the ``Sucrose Density Gradient Centrifugation'' and ``Metrizamide-Sucrose Density Gradient Centrifugation'' sections except that the vesicle or microsome fraction prepared from the antral mucosa containing exogenous [H]DFP-labeled pancreatic trypsin was used as a sample.

Enzymatic Activity Assay

All enzymatic reactions were done at 37 °C. The trypsin-like protease activity was assayed by incubation for 60 min in 200 µl of a reaction mixture containing 0.1 mMt-butyloxycarbonyl(Boc)-Gln-Gly-Arg-MCA, 50 mM Tris-Cl (pH 8.8), and 1 mM CaCl. 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 was measured fluorometrically with an excitation wavelength of 370 nm and an emission wavelength of 460 nm in a Hitachi fluorescence spectrophotometer 650-10S. The pH dependence of activity was measured by the same assay method except that Tris-Cl or bis-Tris-propane buffers of respective pH values were used. Aminopeptidase activity was assayed by the method of Usui et al.(11) with some modifications in 200 µl of a reaction mixture containing 0.1 mM Ala-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's method (12) with 10 mMp-nitrophenyl phosphate in 500 µl of 50 mM sodium acetate buffer (pH 5.4). After the reaction for 1 h, 500 µl of 0.2 N NaOH was added, and the absorbance at 410 nm was measured. Glucose-6-phosphatase activity was assayed by the method described by Swanson(13) , and the amount of liberated inorganic phosphate was determined by the method of Leloir and Cardini(14) . -Mannosidase activity was assayed by the method of Storrie and Madden(15) .

Protein Determination

Protein was determined by measuring the absorbance at 280 nm of the sample solution or by the method of Smith et al.(16) using 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 Trypsin-like Endoprotease from the ER-Microsome

Ten volumes of 1% sodium deoxycholate in 50 mM Tris-Cl (pH 9.0) was added to the pooled active fraction from the density gradient centrifugation and solubilized by magnetic stirring overnight at 4 °C. The solubilized solution was dialyzed against 20 mM Tris-Cl (pH 9.0), including 0.25% sodium deoxycholate and was centrifuged at 100,000 g for 1 h (Beckman 50.2 Ti rotor). The resulting clear supernatant was applied to a DE52 column (2.0 20 cm) equilibrated with 20 mM Tris-Cl buffer (pH 9.0) and 0.1% Lubrol PX, and the column was washed with the same buffer and eluted with a linear gradient of 0-0.5 M of NaCl. To the pooled active fraction was added ammonium sulfate to 30% saturation. This sample was centrifuged for 20 min at 20,000 g, and the supernatant was applied to a butyl-Toyopearl 650 column (2 20 cm) equilibrated with 20 mM Tris-Cl (pH 8.8), 0.02% Lubrol PX containing ammonium sulfate at 30% saturation. The column was washed with the same buffer and then eluted with a decreasing gradient of ammonium sulfate (30 to 0% saturation) in a total volume of 800 ml at a flow rate of 1 ml/min. The active fractions were pooled and brought to 30% ammonium sulfate saturation. This solution was concentrated through a small column of butyl-Toyopearl to 4 ml and then submitted to gel filtration on a Sepharose CL-6B column (2.5 120 cm). Elution was performed with 20 mM Tris-Cl (pH 8.8) containing 0.02% Lubrol PX and 0.2 M NaCl (flow rate, 0.3 ml/min; fraction size, 8 ml). The active fractions were pooled and applied to a benzamidine-Sepharose 6B column (0.8 2.5 cm) and eluted with 20 mM Tris-Cl buffer (pH 8.8) containing 50 mM benzamidine hydrochloride, 0.02% Lubrol PX, and 0.5 M NaCl (flow rate, 0.75 ml/min; fraction size, 1 ml). The active fractions were pooled and dialyzed against 20 mM Tris-Cl buffer (pH 8.8), 0.02% Lubrol PX, and benzamidine was completely removed.

Partial Purification of Trypsin-like Endoprotease from the Vesicle

500 µl each of the active fractions from the metrizamide-sucrose density gradient centrifugation of the vesicle fraction was added to 4.5 ml of 0.02% Lubrol PX solution. Each solution was frozen, thawed, vortexed five times, and centrifuged for 40 min at 100,000 g. Each supernatant fraction and porcine pancreatic trypsin as a control enzyme were analyzed in a butyl-Toyopearl 650 chromatograph in which all the buffer solutions for equilibration, washing, and elution of the column were prepared with or without added 0.1%(w/v) Lubrol PX. The butyl-Toyopearl 650 column (0.8 1.5 cm) was equilibrated with 40 mM Tris-Cl (pH 8.8) containing ammonium sulfate at 30% saturation. The column was washed with the same buffer and then eluted with a decreasing gradient of ammonium sulfate (30 to 0% saturation) in a total volume of 20 ml at a flow rate of 0.75 ml/min. The concentration of ammonium sulfate in each fraction was measured using a refractometer Type N1 (Atago Co.), comparing the Brix percent of each fraction with each standard concentration of ammonium sulfate in the same buffer. The elution profile was compared as a function of the ammonium sulfate concentration at which each enzyme was eluted.

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 8.8), 1 mM CaCl, and 36 fmol of the purified enzyme. The fragments of oligopeptides produced by enzymatic reaction were separated by HPLC with a Hitachi 655A-11/LC5000 system using a TSKgel ODS-120T reverse phase column (0.46 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.

Amino Acid Sequencing

Preparation and sequencing of peptide fragments were performed according to Iwamatsu(17) . The purified enzyme was electroblotted onto polyvinylidene difluoride membranes. The enzyme on polyvinylidene difluoride membranes was reduced and S-carboxymethylated, then incubated with Acromobacter protease I (lysyl endopeptidase). Released peptide fragments were separated by HPLC using a µ-Bondashpere 5µ C8-300Å column (2.1 150 mm, Waters) and submitted to amino acid sequencing with a Shimadzu PSQ-1 gas phase sequencer.


RESULTS

Distribution of Trypsin-like Protease Activity on Sucrose or Metrizamide-Sucrose Density Gradient Centrifugation

Through differential centrifugation, trypsin-like serine protease activities were found both in the microsome fraction and in the vesicle fraction of gastric antral mucosa. Then we used the density gradient centrifugation method to specify the distribution of the trypsin-like activities from both fractions. As shown in Fig. 1, a and b, the trypsin-like protease activity toward Boc-Gln-Gly-Arg-MCA from the microsome fraction was distributed mainly at 1.22-1.23 g/ml, which seemed to be located at the ER fraction as judged from the distribution of glucose-6-phosphatase, an ER marker enzyme (Fig. 1e). The activity of -mannosidase, a Golgi marker enzyme, was broadly distributed in both microsome and vesicle fractions (Fig. 1f). The distribution pattern of the trypsin-like protease activity was different from that of aminopeptidase and acid phosphatase (Fig. 1, b and d). On the other hand, the trypsin-like protease activity from the vesicle fraction was observed in two broad peaks (fractions 6-7 and 13-15 in Fig. 1c), unlike that of the microsome fraction. Its activity was very low as compared with that of the ER-microsome fraction, and its distribution pattern was also different from those of aminopeptidase and acid phosphatase. The trypsin-like protease activity from the vesicle fraction was clearly separated into two peaks (peak a: d = 1.03 g/ml; peak b: d = 1.11-1.12 g/ml) in the subsequent metrizamide-sucrose density gradient centrifugation at which the osmolarity of each gradient step was adjusted to 300 mosm/kg (Fig. 2).


Figure 1: Fractionation of the trypsin-like protease activity from the gastric antral microsome and vesicle fractions through sucrose density gradient centrifugation. a, density () and protein of the microsome (), and vesicle () fractions. b, trypsin-like protease activity () and aminopeptidase activity () of the microsome fraction. c, trypsin-like protease activity () and aminopeptidase activity () of the vesicle fraction. d, acid phosphatase activity of the microsome () and vesicle () fractions. e, glucose-6-phosphatase activity of the microsome () and vesicle () fractions. f, -mannosidase activity of the microsome () and vesicle () fractions.




Figure 2: Fractionation of the trypsin-like protease activity from the gastric antral vesicle fraction by metrizamide-sucrose density gradient centrifugation. , trypsin-like protease activity toward Boc-Gln-Gly-Arg-MCA; , protein; , density.



[H]DFP-labeled pancreatic trypsin was used as the exogenous authentic enzyme to analyze whether the present trypsin-like protease(s) and the exogenous pancreatic trypsin parallel in cellular fractionation of the antral mucosa and density gradient centrifugation of microsome and vesicle fractions. Over 90% of the radioactivity of exogenous [H]DFP-labeled pancreatic trypsin was found in the cytosol fraction upon cellular fractionation of the antral mucosa (data not shown). When the vesicle or microsome fraction, which still retained a minute quantity of the exogenous [H]DFP-labeled pancreatic trypsin, was applied to the sucrose or metrizamide-sucrose density gradient centrifugation, the greater part of the radiolabeled enzyme remained at the original density gradient section on which the vesicle or the microsome samples had been loaded, whereas the gastric tryptic activities from the antral mucosa were distributed in other distinct density regions (Fig. 3).


Figure 3: Density gradient fractionation of the vesicle and microsome fractions with exogenous [H]DFP-labeled pancreatic trypsin. A, metrizamide-sucrose density gradient fractionation of the vesicle fractions from the normal antral mucosa and the mucosa supplemented with exogenous [H]DFP-labeled pancreatic trypsin. B, sucrose density gradient fractionation of the microsome fractions from the normal antral mucosa and the mucosa supplemented with exogenous [H]DFP-labeled pancreatic trypsin. Density gradient fractionations were done as described under ``Experimental Procedures.'' , relative trypsin-like protease activity; , H radioactivity.



Purification of the Trypsin-like Protease from the ER-Microsome Fraction

The solubilization effects of the activity from the pooled ER-microsome fraction obtained above was examined using various detergents. The effects (percentage of solubilization) decreased in the following order: sodium deoxycholate or Triton X-100 (>95%) > sodium cholate (84%) > Lubrol PX (20%). However, Triton X-100 was somewhat inhibitory to the protease activity, so the trypsin-like protease was solubilized with sodium deoxycholate. Then, the enzyme was purified by a series of chromatographic steps on DEAE-cellulose (DE52), butyl-Toyopearl 650, Sepharose CL-6B, and benzamidine-Sepharose 6B as shown in Fig. 4. The protease activity was separated into two peaks: form A (fractions 38-48) and form B(50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62) in butyl-Toyopearl 650 column chromatography (Fig. 4b). Each active fraction was applied to a Sepharose CL-6B column, which gave a single peak of activity for both form A (Fig. 4c) and form B (data not shown). Each pooled active fraction was completely purified by benzamidine-Sepharose 6B column chromatography (Fig. 4d). Thus, 3.6 µg of form A and 2.4 µg of form B were finally isolated from 77 g of wet antral mucosa from five stomachs ().


Figure 4: Purification of the trypsin-like protease from the ER-microsome fraction of gastric antral mucosa. a, DEAE-cellulose (DE52) chromatography. b, TSKgel butyl-Toyopearl 650 chromatography. c, Sepharose CL-6B chromatography (only the result with form A is shown); , trypsin-like activity toward Boc-Gln-Gly-Arg-MCA; , A. d, SDS-PAGE of benzamidine-Sepharose 6B fractions. Fractions 2-6 of each of the forms A and B were applied to 12.5% PAGE in SDS.



Purification of the Trypsin-like Protease from the Vesicle Fraction

When the peaks a and b were solubilized with 0.02% Lubrol PX, the extent of solubilization of the activity peak a was about 50% low as compared with that of the peak b (data not shown). The solubilized fractions of the activity peaks a and b showed some difference on the butyl-Toyopearl chromatography in which all the buffer solutions for equilibration, washing, and elution of the column included 0.1% Lubrol PX (Fig. 5A). The elution profiles of the tryptic activities from peaks a and b were very similar with those of the two forms A and B from the ER-microsome fraction, respectively (Fig. 4b). The trypsin-like protease from peak a was eluted a little earlier (at 21% ammonium sulfate) than that from peak b (at 19%). The two types from peaks a and b were distinctly different from porcine pancreatic trypsin which was eluted at 14% ammonium sulfate, although they showed essentially the same substrate specificity toward MCA substrates with pancreatic trypsin (I). On the other hand, the two forms and the pancreatic trypsin did not show much difference in elution position on butyl-Toyopearl chromatography in which all the buffer solutions for equilibration, washing, and elution of the column were free of Lubrol PX (Fig. 5B), although the two forms a and b were slightly different from that of the pancreatic trypsin. These results indicated that there is some difference in the extent with which Lubrol PX weakens the hydrophobic interactions between the trypsin or trypsin-like proteases and the butyl groups conjugated on the Toyopearl beads.


Figure 5: TSKgel butyl-Toyopearl 650 chromatography of the solubilized fractions of peaks a and b from the vesicle fraction. The enzymes were eluted with a decreasing gradient of ammonium sulfate (30 to 0%) (A) with and (B) without 0.1% (w/v) Lubrol PX, as described under ``Experimental Procedures.'' The concentration of ammonium sulfate in each fraction was represented in the x axis of abscissas, and the trypsin-like protease activities of the solubilized fractions from peaks a and b were assayed as described under ``Experimental Procedures'' and were compared as a function of ammonium sulfate concentration at which each enzyme was eluted. , the solubilized fraction of peak a; , the solubilized fraction of peak b; , porcine pancreatic trypsin (control).



Properties of the Trypsin-like Protease

Both forms A and B showed on 12.5% SDS-PAGE the same molecular weight, 22,400 under nonreducing conditions and 28,000 under reducing conditions (Fig. 6). They showed the optimum activity at pH 8-9 (Fig. 7) and were strongly inhibited by DFP, bovine pancreatic trypsin inhibitor, leupeptin, and benzamidine (). Form A cleaved only on the C-terminal side of Arg or Lys residues in various oligopeptides (Fig. 8). Both forms A and B cleaved the Arg-MCA bond in various MCA substrates, and the rate of cleavage decreased in the order: Boc-Gln-Gly-Arg-MCA > Boc-Gln-Ala-Arg-MCA > Boc-Gln-Arg-Arg-MCA . . . , which coincided with that of porcine pancreatic trypsin (I). Likewise, the two active fractions (peaks a and b) from the metrizamide-sucrose density gradient centrifugation of the vesicle fraction (Fig. 2) showed essentially the same substrate specificity toward these synthetic MCA substrates (I).


Figure 6: SDS-PAGE of the purified trypsin-like protease (form A). The sample was separately applied to 12.5% PAGE in SDS under nonreducing (lane 1) and reducing (lane 2) conditions. The gel, including lanes 1 and 2, was stained by the silver staining method, and the gel of another nonreducing lane was transferred into the routine running buffer without SDS, and SDS was extracted for 20 min. The gel was sliced into pieces of 2-mm width for overnight extraction in 200 µl of the assay buffer. Aliquots of the extracts were assayed as described under ``Experimental Procedures.''




Figure 7: pH dependence of the trypsin-like protease forms A and B. The symbols used are: --, form A/bis-Tris-propane buffer; --, form A/Tris-Cl buffer; - - -- - -, form B/Bis-Tris-propane buffer; - - -- - -, form B/Tris-Cl buffer.




Figure 8: Sites of cleavage by the trypsin-like protease in various peptides. The arrows (▾, ▾, and ) indicate the major, medium, and minor cleavage sites, respectively. The amino acid sequences of peptides are shown in one-letter codes. pE, pyroglutamic acid; -NH, amide.



Partial Amino Acid Sequence of the Trypsin-like Protease

Each of the two forms of the protease was digested separately with Acromobacter protease I (lysyl endopeptidase) and the resulting peptides were separated by HPLC. The HPLC patterns thus obtained were the same for the two forms (data not shown), and five internal fragments from each could be obtained in a pure form and sequenced partially or completely. The N-terminal and five internal sequences determined for both forms were identical with those of porcine pancreatic trypsin (18) as shown in . Furthermore, the N-terminal residue of each of the five internal sequences is presumed to be preceded by a lysine residue in the original protein as judged from the specificity of Acromobacter protease I(19) . This is also consistent with the sequence of pancreatic trypsin(18) .


DISCUSSION

In this study, trypsin-like serine protease activities were found in the ER-microsome fraction and also in the vesicle fraction of the gastric antral mucosa, and two forms of the trypsin-like protease were purified from the ER-microsome fraction. They were separated from each other by butyl-Toyopearl chromatography, but were indistinguishable in molecular size, substrate specificity, pH dependence of activity, and the effects of inhibitors. Surprisingly, the N-terminal and partial internal sequences, including 59 residues, of both forms were found to be identical with those of porcine pancreatic trypsin ().

Comparison of the partial sequences of the present enzyme with the corresponding sequences of some pancreatic trypsins from other species showed that the identity is 78% with bovine trypsin(20) , 68% with rat trypsin(21) , and 66% with human trypsin(22) . Furthermore, these differences roughly corresponded to those for the entire amino acid sequences among these trypsins; the identity of porcine trypsin is 79% with bovine trypsin, 79% with rat trypsin, and 78% with human trypsin. On the other hand, the identities with other typical serine proteases were 36% with bovine chymotrypsin(23, 24, 25, 26) , 34% with porcine (27) and bovine (28) enteropeptidases, human factor XIa(29) , and porcine elastase(30) , 33% with dog tryptase(31) , 31% with human plasma kallikrein(32) , and 27% with human hepsin(33) . Considering these results, especially the fact that the present enzyme showed differences of over 20% in amino acid sequence identity even with the bovine, rat, and human pancreatic trypsins, the identity of the partial sequence, including 59 residues, of the present enzyme with that of porcine pancreatic trypsin (223 residues) seems to permit us to conclude, beyond statistical consideration, that trypsin (or trypsin variant, an enzyme almost identical with trypsin) has been found in the stomach mucosa.

The trypsin activities from the ER-microsome fraction and that from the vesicle fraction were found at different densities in sucrose density gradient centrifugation. However, the two peaks of trypsin-like activity from the vesicle fraction were also indistinguishable from each other and from the enzyme of the microsome fraction in substrate specificity (I) and pH dependence of activity and the effects of inhibitors (data not shown). Two types of trypsin-like proteases (peaks a and b in Fig. 2) from the vesicle fraction were differently eluted from butyl-Toyopearl 650 in which proteins are separated mainly by differences in hydrophobic binding forces (Fig. 5A) and its elution profile seemed to be consistent with each of the two forms A and B from the ER-microsome (Fig. 4b). This indicates the possibility that the same gastric trypsin may be separately associated with different organelle membranes. It may be possible that some of the trypsin in the ER could move, through Golgi, to the vesicle fraction. Thus, the two activity peaks of the vesicle fraction may correspond to the two enzyme forms of the ER-microsomes. It seems interesting to elucidate how the enzymes can associate with the ER-microsome or vesicle. Compared with pancreatic trypsin, they may have some minor variation in the amino acid sequence that increase the affinity to the membranes.

Trypsin is normally synthesized in the -cells of the pancreas as trypsinogen, which is then secreted into duodenum and activated by enteropeptidase and/or trypsin, and functions as a digestive protease in duodenum and intestine(34, 35) . Therefore, the possibility of contamination of pancreatic trypsin due to duodenogastric reflux may be considered. To investigate this possibility, an exogenous authentic enzyme, [H]DFP-labeled pancreatic trypsin, was added to antral mucosa, then homogenization, cellular fractionation, and density gradient fractionation were done. Metrizamide-sucrose or sucrose density gradient fractionation of the vesicle or microsome fractions which still included a minute quantity of exogenous [H]DFP-labeled pancreatic trypsin showed that the exogenous trypsin was retained at the original density gradient region where the sample had been applied, but that the gastric tryptic activities from the vesicle or ER-microsome source were distributed at other distinct density regions (Fig. 3). These results exclude the possibility of contamination of pancreatic trypsin from duodenum.

Davis et al.(36) reported that the trypsin mRNA was found in the stomach mucosa of both normal and transgenic mice. They showed that the expression of pancreatic genes, including the trypsin genes, is controlled differently in stomach and in pancreas and excluded the possibility of pancreatic contamination. On the other hand, a trypsin-like protease complexed with -macroglobulin was found in porcine gastric mucosa in our previous study(4) . More recently, the possibility that trypsin may function in other normal tissues besides in duodenal and intestinal tracts has been suggested by Wiegand et al.(37) . They reported the cloning of the cDNA encoding two forms of human brain trypsinogen which show 99.2% homology with that of human pancreatic trypsinogen III (22) in the sequences of exon 2-5 regions. Furthermore, some papers have reported that pancreatic trypsin inhibitor was found in normal gastric mucosa (7-9). Protease inhibitors such as -macroglobulin and pancreatic trypsin inhibitor in gastric mucosa might also be involved in regulation of the trypsin-like activity, whereas they might also play a role, if secreted, in gastric mucosal defense by inhibiting duodenal proteases upon duodenogastric reflux. However, the occurrence of the gastric trypsin in the ER-microsome fraction and the presence of trypsin mRNA in the glandular portion of the stomach (36) support our idea that gastric trypsin (or trypsin variant) may be produced in the gastric mucosal cell and function as an intracellular protease in the gastric mucosal tissue. It may function as a specific degrading or processing enzyme in the ER or vesicles of gastric mucosal cells. The gastric trypsin is thought to be synthesized as trypsinogen like pancreatic trypsin, but its conversion to trypsin seems to occur intracellularly unlike pancreatic trypsin. It will be interesting to investigate whether the gastric trypsinogen is activated by the action of an enteropeptidase or an enteropeptidase-like enzyme which might be present intracellularly in gastric mucosa or by another intracellular protease. Further enzymatic and molecular characterization, including the investigation of the structural relationship among the isoforms, and elucidation of the roles of the gastric trypsin and the activation mechanism of its proform, remain to be done in future studies.

  
Table: Purification of the trypsin-like protease from the microsomal fraction of porcine gastric antral mucosa


  
Table: Effects of inhibitors and metal ions on Boc-Gln-Gly-Arg-MCA hydrolyzing activity of the trypsin-like protease from the microsome fraction


  
Table: Substrate specificity toward MCA substrates of the trypsin-like proteases from the ER-microsome and vesicle fractions


  
Table: 0p4in Peptides were obtained by digestion with Acromobacter lysyl endopeptidase.(119)


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.

§
Present address: School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan.

To whom correspondence should be addressed. Present address: School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. Tel.: 81-426-76-7146, Fax: 81-426-76-7149.

The abbreviations used are: VIP, vasoactive intestinal polypeptide; ER, endoplasmic reticulum; MCA, 4-methylcoumaryl-7-amide; BAM-12P, bovine adrenal medulla dodecapeptide; E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane; BCA, bicinchoninic acid; DFP, diisopropyl fluorophosphate; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; TLCK, N-tosyl-L-lysine chloromethyl ketone; PMSF, phenylmethanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; Boc, t-butyloxycarbonyl; AMC, 7-amino-4-methylcoumarin; Bz, benzoyl; Suc, succinyl; HPLC, high performance liquid chromatography; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.


ACKNOWLEDGEMENTS

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


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