From the
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.
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),
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.
Other reagents used were of the highest grade
available.
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
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
We thank Dr. Hideshi Inoue and Yuichi Tsuchiya for
valuable discussions on this work and Yasuko Sakurai for amino acid
analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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.
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.
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.
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) .
-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.
-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)
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.