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/ . Biochem. 93, 743-754 (1983)
Occurrence of Two Different Pathways in the Activation
of Porcine Pepsinogen to Pepsin1
Takashi KAGEYAMA and Kenji TAKAHASHI
Received for publication, September 9, 1982
Activation of porcine pepsinogen at pH 2.0 was found to proceed simultaneously
by two different pathways. One pathway is the direct conversion process of pepsinogen to pepsin, releasing the intact activation segment. The isolation of the
released 44-residue segment was direct evidence of this one-step process. At pH 5.5
the segment bound tightly to pepsin to form a 1 : 1 pepsin-activation segment
complex, which was chromatographically indistinguishable from pepsinogen. The
other is a stepwise-activating or sequential pathway, in which pepsinogen is activated
to pepsin through intermediate forms, releasing activation peptides stepwisely.
These intermediate forms were isolated and characterized. The major intermediate
form was shown to be generated by removal of the amino-terminal 16 residues
from pepsinogen. The released peptide mixture was composed of two major peptides comprising residues 1-16 and 17^t4, and hence the stepwise-activating process
was deduced to be mainly a two-step process.
Pepsinogen is converted to pepsin under acidic
conditions. The reaction proceeds autocatalytically, releasing the so-called activation peptides
from the amino(N)-terminal part of the pepsinogen
molecule (7). In porcine pepsinogen, these activation peptides are derived from the N-terminal
44-residue segment (2-4). This activation follows
predominantly an intramolecular mechanism below
pH 3 (5-9). Essentially two reaction pathways
are possible for the activation; i.e., the direct
conversion pathway and the sequential conversion
pathway. In porcine pepsinogen, evidence supporting the latter pathway has been presented
by several investigators. Dykes and Kay reported
that in the presence of pepstatin, a potent inhibitor
of pepsin, the N-terminal 16-residue peptide (residues 1-16) was released first, suggesting the sequential release of the activation segment (10).
They obtained similar results using bovine, chicken,
and canine pepsinogens and bovine prochymosin
(11). We also isolated an intermediate form2
(pseudopepsin) between pepsinogen and pepsin
1
2
This study was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education,
Science and Culture of Japan.
Abbreviations: N,amino; C,carboxyl; SDS, sodium
dodecyl sulfate; SP, sulfopropyl.
Vol. 93, No. 3, 1983
The term 'intermediate form' used in the present paper
and our previous papers (12, 14) differs from the conformational intermediates d, 6, and 4> of Marciniszyn
et al. (9), and corresponds to the pseudopepsin presumed
by Dykes and Kay (10) and Christensen et al. (13).
743
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Department of Biochemistry, Primate Research Institute,
Kyoto University, Inuyama, Aichi 484
744
T. KAGEYAMA and K. TAKAHASHT
upon activation of human pepsinogen in the presence of pepstatin {12). Further, Christensen ei al.
reported that the initial cleavage of porcine pep16
17
MATERIALS AND METHODS
Materials—Porcine pepsinogen (grade I, chromatographically prepared free from pepsin) was
purchased from Sigma. A small amount of minor
components present in the preparation was chromatographically removed prior to use. DEAEToyopearl was obtained from Toyo Soda Manufacturing Co., Tokyo. Sephadex G-50 and SP
(sulfopropyl)-Sephadex were purchased from Pharmacia, fluorescamine from Japan Roche Co.,
Tokyo, and reagents for amino acid sequence
determination from Wako Pure Chem. Ind.,
Tokyo. Carboxypeptidase Y was purchased from
Oriental Yeast Co., Tokyo. Pepstatin was a
Purification of Activation Peptides—The lyophilized activation mixture was dissolved in about
5 ml of 0.1 M sodium acetate buffer, pH 5.5, containing 8 M urea, and subjected to gel filtration on
a column (1.6 x 150 cm) of Sephadex G-50 in the
same buffer. The activation peptides were fractionated into a few peaks separated from the
protein. Fractions containing a peptide mixture
were further purified by chromatography on a
column (1.6x40 cm) of SP-Sephadex in 0 . 1 M
sodium acetate buffer, pH 5.5, containing 8 M
urea. Peptides were eluted with a linear gradient
of NaCl from 0 to 0.75 M using two 300-ml chambers. To remove urea, the pooled fraction was
diluted about 20-fold with 0.1 M sodium acetate
/. Biochem.
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sinogen occurred at the peptide bond Leu-Ile on
activation without pepstatin, forming the intermediate form (pseudopepsin) (13). These results
indicate that porcine pepsinogen is activated to
pepsin through intermediate form(s) by sequential
release of the activation peptides.
However, quite recently, we isolated the intact
activation segment upon activation of Japanese
monkey pepsinogen, indicating that one-step activation occurred exclusively (14). Our results
also showed that the intermediate species was
formed only in the presence of pepstatin. These
results for Japanese monkey pepsinogen strongly
suggested the occurrence of direct conversion of
pepsinogen to pepsin, which differed greatly from
those obtained for porcine pepsinogen. We decided to clarify the activation pathways in porcine
pepsinogen based on the structural evidence and
compare them to those of Japanese monkey pepsinogen.
In the present study, porcine pepsinogen was
activated in solution in the absence of pepstatin,
and both the released peptides and the intermediate protein species were isolated and characterized. The released peptides were shown to
include the intact activation segment of residues
1-44 together with peptides of residues 1-16 and
17-44. These results demonstrate that one-step
activation occurs in porcine pepsinogen along with
the sequential process. A preliminary report on
part of this study has appeared elsewhere (75).
generous gift from Drs. H. Umezawa and T.
Aoyagi. All other chemicals were of reagent
grade.
Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Disc Gel Electrophoresis—The procedure
was similar to that described by Weber and Osborn
(16).
Fluorometric Determination of Peptides—The
peptide concentration was determined by the
fluorometric method using fluorescamine according
to de Bernardo et al. (17). The fluorescence was
measured with a Hitachi Model 203 spectrofluorometer with excitation at 390 nm and emission at
475 nm, using leucine as a standard.
Activation of Pepsinogen—Pepsinogen (10-100
mg) was dissolved in 50 ml of 0.01 M sodium
phosphate buffer, pH 7.0, and the solution was
acidified to pH 2.0 by the addition of 12.5 ml of
0.1 N HC1. The reaction mixture was prepared
and incubated with gentle stirring at 14°C. At
desired intervals, aliquots were withdrawn to examine the extent of activation by assaying the
remaining potential pepsin activity and by SDSdisc gel electrophoresis (for these methods, see the
legend to Fig. 1). The activation was terminated
by the addition of 1 M NH4OH to a final concentration of 0.2 M. The mixture was immediately
frozen, lyophilized, and subjected to gel filtration.
To isolate the resulting protein species, the activation reaction was stopped by raising the pH to
near 5.5 by adding 2.5 ml of 5 M sodium acetate
buffer, pH 5.5, containing an about 3-fold molar
excess of pepstatin over the initial amount of
pepsinogen used. This preparation was subjected
to chromatography on DEAE-Toyopearl.
ACTIVATION OF PORCINE PEPSINOGEN
Vol. 93, No. 3, 1983
RESULTS
Analysis of the Time Course of Pepsinogen
Activation—Pepsinogen was activated at various
concentrations at pH 2.0, and the activation process was analyzed by SDS-disc gel electrophoresis
(Fig. 1). In all cases, pepsinogen disappeared
rapidly after acidification. The resulting protein
species were detected as two bands; one of them
had the same molecular weight as the authentic
pepsin and the other hand a molecular weight
intermediate between those of pepsinogen and
pepsin. The intermediate form was relatively
stable as compared with pepsinogen, but was
gradually converted to pepsin during a long period
of incubation. When analyzed by the proteolytic
activity assay, the activation appeared to be complete within a few min (Fig. 2). The formation
of the intermediate form became predominant
when the initial pepsinogen concentration decreased. Released peptides were also detected as
two bands. The amount of peptide in the high
molecular weight peptide band appeared to be
maximum at I or 2 min and to decrease rather
rapidly, while that in the low molecular weight
peptide band appeared to increase gradually with
the progress of incubation time. However, when
the initial pepsinogen concentration was 1.6 mg/
ml, both peptide bands were scarcely detectable
after 2 min. This may be due to further cleavage
of these peptides to smaller peptides, which were
not retained in the gel.
The activation experiments described in the
following sections were carried out at an initial
pepsinogen concentration of 0.16 mg/ml in the
activation mixture.
Isolation and Characterization of Activation
Peptides—Peptides released after 2-min and 30-min
activation were isolated and characterized. The
lyophilized reaction products were fractionated by
Sephadex G-50 gel filtration (Fig. 3). The protein
mixture was eluted near the void volume, separated from peptides. Peptides in the 2-min activation mixture were separated into 3 peaks (Fractions
1, II, and III). Each fraction showed a single
N-terminal amino acid, and Fractions IF and 11 f
gave a single spot on thin layer electrophoresis
at pH 3.5. These results indicated that each fraction was composed of a single peptide. Electro-
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016
buffer, pH 5.5, and then applied to a column
(1.2x2.5 cm) of SP-Sephadex equilibrated with
the same buffer. After the column was washed
with the buffer, the peptide was eluted with the
same buffer containing 1 M NaCI. The peptide
was freed from the salts by passage of the solution
through a column (1.6x45 cm) of Sephadex G-15
equilibrated with 1 % acetic acid. Purity of each
peptide fraction was examined by N-terminal
amino acid analysis by dansylation (18), and further by electrophoresis on a thin-layer plate (Precoated TLC plate, Cellulose, Merck Co.) at pH
3.5 in pyridine-acetic acid-water ( 1 : 1 0 : 90, by
volume) at 1,000 volts/20 cm for 40min.
Isolation of Pepsinogen, Pepsin, and the Intermediate Form—The activation mixture adjusted to
pH 5.5 was applied to a column (1.15x25 cm) of
DEAE-Toyopearl previously equilibrated with
0.1 M sodium acetate buffer, pH 5.5, containing
7 //M pepstatin. The adsorbed protein was eluted
with a linear gradient of NaCI from 0 to 0.5 M
using two 300-ml chambers followed by 100 ml
of 0.5 M NaCI in the same buffer.
Amino Acid Analysis—Samples for analysis
were hydrolyzed with 1 ml of 6 N HCI at 110°C
for 24 and 72 h in evacuated sealed tubes. The
amino acids were determined with a Hitachi Model
835 amino acid analyzer essentially according to
Spackman et al. (19).
Amino Acid Sequence Determination—The
amino acid sequences of the N-terminal regions of
pepsinogen, pepsin, and the intermediate forms
were determined by a modification (20) of the
manual Edman degradation method (21) using
0.5-1 mg of each protein. Phenylthiohydantoin
derivatives of amino acids were identified by thin
layer chromatography according to Kulbe (22),
and/or high performance liquid chromatography
using a Toyo Soda column LS410K. according to
Omichi et al. (23). The carboxyl(C)-terminal amino
acid sequence of the activation segment was analyzed with carboxypeptidase Y as follows. Two
nmoles of each activation segment was incubated
at 37°C with 20 //g of carboxypeptidase Y in 300
/i\ of 0.1 M sodium phosphate buffer, pH 6.5, containing 10% methanol. Aliquots were withdrawn
at desired times and released amino acids were
determined with the amino acid analyzer.
745
ACTIVATION OF PORCINE PEPSINOGEN
747
200 —
100 —
0
5
TIME OF ACTIVATION (min)
by SP-Sephadex chromatography. Amino acid
analysis showed that it was identical with peptide
1-16 in Fraction III. Fraction VI contained one
major peptide as shown by thin layer electrophoresis, and it was purified by preparative electrophoresis on Whatman 3 MM filter paper. It was
composed of 8 residues corresponding to residues
17-24 of the activation segment. Fractions No.
110-120 (Fig. 3b) contained small peptides and/or
free amino acids and they were not purified further.
Isolation and Characterization of Pepsinogen,
Pepsin, and Intermediate Forms—The activation
mixtures were analyzed by chromatography on
DEAE-Toyopearl in the presence of pepstatin.
After activation for 2 min, several peaks (Fractions A through F) appeared (Fig. 5b). Fraction
B was eluted at the same position as that of
authentic pepsinogen. This was confirmed by
cochromatography of authentic pepsinogen and
the activation mixtre. Upon further incubation
until 30 min, Fractions A and B disappeared and
the relative contents of Fractions E and F increased
as shown in Fig. 5c as Fractions J and K. The
amino acid compositions of some of these fractions
are shown in Table II. Fractions A and B had
Vol. 93, No. 3, 1983
200 —
Q.
0.
100 —
40
60
FRACTION
80
100
NUMBER
Fig. 3. Gel filtration of the activation mixture. Activation' was carried out for 2 min (a) and 30 min (b), and
stopped by the addition of NH4OH. The mixture was
lyophilized, redissolved and subjected to gel filtration.
A column (1.6 x 150 cm) of Sephadex G-50 was equilibrated and eluted with 0.1 M sodium acetate buffer,
pH 5.5, containing 8 M urea. The fraction size was
3 ml. BD andǤalt indicate the positions of blue dextran
and inorganic^alts, respectively. The fractions under
the bars were pooled.
- 100
120
FRACTION
140
NUMBER
Fig. 4. SP-Sephadex chromatography of peptide fraction IV. The column (1.6x40 cm) was equilibrated
with 0 . 1 M sodium acetate buffer, pH 5.5, containing
8 M urea. Peptides were eluted with a linear gradient'of
NaCl in the same buffer. The fractions under the bars
were pooled. The fraction size was 3 ml.
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016
Fig. 2. Time course of activation of porcine pepsinogen
analyzed by the proteolytic activity assay. Activation
was carried out at pH 2.0 and 14°C. The initial concentration of pepsinogen in the activation mixture was
0.16mg/ml. Aliquots of the incubation mixture were
withdrawn at desired intervals, diluted 10-fold with
0.01 M sodium acetate buffer, pH 5.5, and used partly
for the assay of the total proteolytic activity at pH 2.0
and 37°C with bovine hemoglobin as a substrate according to Anson (27). For the assay of the residual pepsinogen, aliquots of the diluted reaction mixture were
mixed with an equal volume of 0.1 M Tris, and incubated
at 14°C for 30 min before the assay to inactivate pepsin
formed. The extent of activation was calculated from
the difference in the activities determined in these two
assays.
748
T. KAGEYAMA and K. TAKAHASHI
TABLE I. Amino acid compositions of purified peptide fractions.1
Number of residues per molecule of peptide t>
Amino acid
II
in
IV-2
IV-3
V
VI
Lys
9.3 (9)
5.9 (6)
2.9 (3)
5.6 (6)
5.9 (6)
2.7 (3)
2. 4 (3)
His
1.8 (2)
1.8 (2)
1.8 (2)
2. 1 (2)
Arg
2.2 (2)
Asp
4.0 (4)
3.0 (3)
3.0 (3)
3.0 (3)
1.8 (2)
1.0 (1)
2. 0 (2)
Thr
0.9 (1)
Ser
0.9 (1)
1.8 (2)
2.0 (2)
0.9 (1)
0.9 (1)
0.9 (1)
1.0 (1)
Glu
1.0 (1)
1.1 (1)
0.9 (1)
1.0 (1)
1. 1 (1)
1. 1 (1)
Pro
2.5 (3)
1.6 (2)
0.7 (1)
1.4 (2) c
1.8 (2)
Gly
1.5 (1)0
1.3 (1)
Ala
3.5 (4)
3.9 (4)
1.3 (1)
3.5 (4)
1. 1 (1)
2. 1 (2)
Val
3.1 (3)
He
Leu
1.1 (1)
7.4 (7)
0.9 (1)
3.1 (3)
0.8 (1)
3.3 (3)
1.0 (1)
2.3 (2)
Tyr
0.2 ( l ) e
Phe
1.8 (2)
0.8 (1)
1.9 (2)
0.1 (I) 8
1.6 (2)
0.2 (l) e
1.8 (2)
Total
N-Terminus f
Yield «(%)
1.8 (2)
1.0 (1)
0. 9 (1)
3.1 (3)
2.5 (3)
3.9 (4)
0.9 (1)
1.1 (1)
0.7 (1)
3.9 (4)
0. 7 (1)
0. 9 (1)
44
28
16
28
25
16
8
Leu
lie
24
Leu
He
He
Leu
nd
78
5
28
87
nd
10
a The composition of Fraction IV-1 is not shown, since this fraction was a mixture of a few peptides as clarified by
N-terminal analysis. The composition of the high molecular weight peptide isolated from Fraction B after
DEAE-Toyopearl chromatography was the same as that of Fraction I. i> The values were calculated by assuming
the number of aspartic acids to be 4.0 in Fraction I, 3.0 in Fractions II, IV-2, and IV-3, 2.0 in Fraction VI, and 1.0
in Fractions III and V. <= Assumed as 2 residues. « Assumed as 1 residue. • Assumed value allowing for loss
during acid hydrolysis. ' Determined by dansylation according to Gray and Hartley (75). s Yields of Fractions
I, II, III, and V were calculated after Sephadex G-50 gel filtration, and those of Fractions IV-2 and IV-3 were calculated after SP-Sephadex chromatography. These values were based on the amounts of peptides determined by
amino acid analysis, nd: Not determined.
practically the same composition as pepsinogen,
and Fractions E, F, J, and K the same composition as pepsin (data for Fractions J and K not
shown). The compositions of Fractions C, D,
G, H, and I were intermediate between those of
pepsinogen and pepsin, and those of Fractions C,
D , G, and I were nearly identical with one another
(data for Fractions G and I not shown). The
somewhat lower lysine value for Fraction D may
be due to contamination of the pepsin fraction
{i.e., Fraction E).
The N-terminal sequences of these fractions
were determined by manual Edman degradation
(Table III). Analysis of the N-terminal 5-residue
sequence of Fraction B indicated that this fraction
contained the N-terminal sequences of both pepsinogen and pepsin. Moreover, two protein bands
corresponding to pepsinogen and pepsin, and one
peptide band corresponding to the high molecular
weight peptide were detected in Fraction B by
SDS-disc gel electrophoresis (Fig. 6). The peptide
was isolated from Fraction B by adsorption with
SP-Sephadex in the presence of 8 M urea. Amino
acid analysis and N-terminal analysis showed that
the peptide was identical with the 44-residue activation segment (see footnote of Table I). From
these results Fraction B was judged to be a mixture of pepsinogen and a 1 : 1 complex of pepsin
/ . Biochem.
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I
ACTIVATION OF PORCINE PEPSINOGEN
749
TABLE II. Amino acid compositions of pepsinogens, pepsins, and the intermediate forms purified by DEAEToyopearl chromatography."
Number of residues per molecule of protein b
Amino acid
B
A
C
F
E
D
H[
Pgc 11 P»
Lys
11.4 (11)
9.5 (10)
6.7 ( 7)
5.4 ( 5)
1.3 ( 1)
0.9 ( 1)
4.7 ( 5 )
10
7
1
His
2.8 ( 3)
2.2 ( 2)
2.5 ( 3)
2.0 ( 2 )
0.9 ( 1)
0.8 ( 1)
1.8 ( 2 )
3
3
1
Arg
3.6 ( 4 )
3.7 ( 4)
4
2
2
46 45
42
46.0 (46)
46.0 (46)
Thr
28. K (28)
28.8 f (29)
27.9 f (28)
27.9 f (28)
Ser
43.5 f (44) 44. 3'(44)
44.7'(45)
45.5 f (46) 43.7 f (44) 45. If (45)' 45.7f(46)
46 44
44
Glu
27.0 (27)
28.2 (28)
27.7 (28)
28.9 (29)
27.5 (28)
27.3 (27)
27.5 (28)
28
27
26
Pro
17.2 (17)
18.2 (18)
17.7 (18)
15.7 (16)
18.3 (18)
18
17
15
26.6 f (27) 27.4f(27)
28. Of (28) 28 27 27
Gly
37.3 (37)
35.5 (36)
17.0 (17)
35.0 (35)
36.3 (36)
33.9 (34)
15.2 (15)
34.6 (35)
34.1 (34)
36
36
35
Ala
21.8 (22)
19.4 (19)
20.7 (21)
20.7 (21)
18.8 (19)
18.0 (18)
21.6 (22)
20 20
16
Val
24.3 (24)
25.0 (25)
23.6 (24)
24.3 (24)
23.7 (24)
23.9 (24)
23.9 (24)
25
22
22
2.3 ( 2)
2.6 ( 3)
2.5 ( 3)
2.6 ( 3)
2.7 ( 3)
2.2 ( 2)
4
4
4
lie
27.2 (27)
27.0 (27)
26.2 (26)
26.6 (27)
25.3 (25)
25.5 (26)
24.7 (25)
26 26
25
Leu
32. 1 (32)
Met
nd
32.6 (33)
29.4 (29)
29.7 (30)
27.3 (27)
26.7 (27)
28. 1 (28)
34 29
27
Tyr
nd
15. 1 (15)
15.5 (16)
16.2 (16)
15.5 (16)
14.4 (14)
16
16
15
Phe
nd
17.1 (17)
16.3 (16)
17.6 (18)
15.8 (16)
15.1 (15)
16.3 (16)
16.3 (16)
16
16
14
Yield g(%)
1.7
10.3
13.5
Relative yield (%)
•»,»—
23
8.4
1
13.3
5.7
-v*'—
•v^-—
41
36
6.6
a A through H indicate the fractions purified by DEAE-Toyopearl chromatography. The compositions of Fractions G, 1, J, and K are not shown, since they are essentially the same as those of Fractions C, D, E, and F, respectively. b The values were calculated by assuming the number of aspartic acids to be 46.0 in Fractions A and B,
45.0 in Fractions C and D, 43.0 in Fraction H, and 42.0 in Fractions E and F. Except for serine and threonine,
each value is an average of values obtained for 24- and 72-h hydrolysis. The values in parentheses are nearest
integers. Half-cystine and tryptophan were not determined. The compositions of the intermediate species and
pepsin may include one alanine and two valine residues per molecule derived from the bound pepstatin. c Composition of pepsinogen from the sequence of the activation segment (2-4) and pepsin (26). i Composition of the
intermediate form expected from residues 17-370 of pepsinogen. « Composition of pepsin from the known amino
acid sequence (26). f Values extrapolated to zero time of hydrolysis, e Yield after DEAE-Toyopearl chromatography. The yields of Fractions G, I, J, and K were 10.4, 10.4, 24.4, and 8.5 percent, respectively, nd: Not determined.
and the activation segment. This was also supported by the fact that Fraction B had the same
elution position and amino acid composition as
authentic pepsinogen as previously mentioned.
On quantitative determination of the N-terminal
residues in Fraction B, the complex was estimated
to occupy about 5 0 % of Fraction B. These
results indicate that the activation segment formed
Vol. 93, No. 3, 1983
a tight complex with pepsin at pH 5.5 and that
the complex cochromatographed with pepsinogen
at the same pH. When the activation was terminated by raising the pH to 5.5 and the activation
mixture was chromatographed in the absence of
pepstatin, almost all the intermediate forms (Fractions C, D, G, and 1) were converted to corresponding pepsins during chromatography, whereas
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Asp
2.0 ( 2)
2.1 ( 2 )
1.9 ( 2)
1.9 ( 2)
2.0 ( 2)
45.0 (45) 45.0 (45) 42.0 (42) 42.0 (42) 43.0 (43)
ACTIVATION OF PORCINE PEPSINOGEN
751
10
20
30
40
LVKVPLVRKKSLRQNLIKNGKLKDFLKTHKHNPASKYFPEAAALIGDEP
pepsinogen (Pg)
:
-intermediate (C,D,G,I ) \ -intermediate'(H)
'
-activation
segment ( I ) -
-m,v-
IV3
contained at least 2 isozymes which were chromatographically separable from each other after
the activation segment was released completely or
partially. The molar ratio of these two isozymes
was estimated to be about 7 : 3. Although Fraction H was contaminated by Fractions G and I,
the major component was shown to have the
N-terminal sequence :Phe-Leu-Lys-. Fraction H
is therefore thought to have been generated by
removal of the N-terminal 24-residues from pepsinogen. The sequence of Fraction A could not
be determined because of the small amount.
Assignment of Peptides and Proteins, and the
Ratio of Two Activation Pathways—Figure 7 shows
the assignment of the released peptides and the
protein species isolated and identified. Isolation
and characterization of the 44-residue activation
segment together with shorter activation peptides
as well as the intermediate protein species from
the activation mixture of pepsinogen clearly
showed that porcine pepsinogen is largely activated simultaneously by two different pathways,
i.e., one-step activation with initial cleavage at the
44
45
Leu-Ile bond and a stepwise-activating process
16
17
with first cleavage at the Leu-Ile bond, followed
44
45
by cleavage at the Leu-Ile bond.
The yields of released peptides and resulting
protein species are shown in Tables I and II,
respectively. The yield of the 44-residue activation
segment was about 10% after 2-min activation.
This value appears to be lower than expected from
the proportion of the one-step activation pathway,
since the 44-residue segment was cleaved to smaller
Vol. 93, No. 3, 1983
peptides rather rapidly (Fig. 1). Assuming that
in the early period of activation the 28-residue
peptide was formed exclusively by the cleavage
of the 44-residue segment released, the proportion
of the one-step pathway could be estimated to be
maximally about 40%. This assumption was
based on the results in Fig. 1, which showed the
intermediate form to be rather stable when formed
and its further conversion to pepsin by release of
the 28-residue peptide proceeded gradually. On
the other hand, this relatively stable character of
the intermediate form enabled us to estimate
directly the proportion of the sequential pathway
from the relative yields of resulting protein species
(i.e., pepsin, intermediate forms and the complex).
At 2-min activation, the value was about 46%.
These results indicate that under the present activation conditions the two activation pathways
operated nearly equally.
DISCUSSION
As described in the preceding section, porcine pepsinogen was shown to be activated to pepsin at pH
2.0 simultaneously through two different pathways.
These are schematically illustrated in Fig. 8. The
activation reaction at this pH should proceed predominantly intramolecularly as shown by several
investigators (5-7). The occurrence of the onestep process, in which the intact activation segment
of 44 residues is released directly from porcine
pepsinogen, had not been reported previously.
Therefore the isolation, in this study, of this
intact activation segment from the activation mixture of pepsinogen is the first direct evidence of
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016
Fig. 7. Assignment of protein species and various peptides obtained on activation of pepsinogen
for 2min and 30min. The symbols are the same as those in Figs. 3, 4, and 5. Fraction I
corresponds to the high molecular weight peptide band, and Fractions II to VI correspond to
the low molecular weight peptide band in Fig. 1.
T. KAGEYAMA and K. TAKAHASHI
752
\pH5.5
\
X
pepsin-activation
• segment complex
N
—t
this conversion in the activation of porcine pepsinogen. As the initial pepsinogen concentration
in the activation mixture was increased, the proportion of the one-step pathway seemed to increase. This appears to indicate that the one-step
pathway may be partly due to the intermolecular
action of the pepsin formed, although the intramolecular mechanism is thought to be predominant at pH 2. At pH 2 the activation segment
was further cleaved into smaller peptides rather
rapidly. At pH 5.5, however, it formed a tight
complex with pepsin and the complex behaved
chromatographically like pepsinogen. The activation segment could not be removed from the complex even by adsorption to a cation exchange
resin such as SP-Sephadex but it could finally be
released from the complex by denaturation of the
complex with 8 M urea after alkali treatment.
These results seem to indicate that the 44-residue
segment has a much higher affinity to pepsin than
other shorter activation peptides including the Nterminal 16-residue peptide which was reported to
have a Kx value of 5.7 x lO"8 M (24) or 2.5 x 10~8 M
(25).
Previously, Dykes and Kay could obtain only
the N-terminal 16-residue peptide from an activa-
tion mixture upon activation of porcine pepsinogen
at pH 2.5 in the presence of pepstatin, suggesting
that the occurrence of a one-step process was
unlikely (10). It may be possible, however, that
the presence of pepstatin somewhat altered the
course of the activation reaction. The failure in
obtaining the intact activation segment may also
be due to the rather short life span of the activation segment around pH 2 and the high affinity of
the segment to pepsin around pH 5.5.
The other type of activation pathway is a
sequential or stepwise-activating process, in which
activation proceeds through intermediate forms.
In the present study, the intermediate forms (Fractions C, D, G, and I) were generated by removal
of the N-terminal 16-residue peptide from pepsinogen. These intermediate forms are thought
to be the same protein presumed by Dykes and
Kay (10) and Christensen et al. (13) to be pseudopepsin. A part of these intermediate forms was
converted to another intermediate (Fraction H),
which was formed by releasing further an 8-residue
peptide from the N-terminal region. The major
part of the intermediate forms (Fractions C, D,
G, and I), however, is though to be converted to
pepsin directly, since only a small amount of
/ . Biochem.
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intermediate
Fig. 8. The proposed activation process of porcine pepsinogen at pH 2.0.
A pepsinogen molecule at neutral pH is expressed as a square form with an
unreleased activation segment and an undeveloped active site. A pepsinogen
molecule after a conformational change at acidic pH, molecules of the intermediate forms, and a pepsin molecule are expressed as circular forms with a
developed active site. The dashed double-lined arrow indicates the intermolecular attack of pepsin formed against pepsinogen.
ACTIVATION OF PORCINE PEPSINOGEN
44
45
segment to pepsin: i.e., Leu-Ile (porcine pepsino47
48
gen) and Leu-Ile (Japanese monkey pepsinogen),
and the bond in the middle region of the activation
16
17
segment: i.e., Leu-Ile (porcine pepsinogen) and
25
26
Asp-Phe (Japanese monkey pepsinogen). This suggests that these regions may be located structurally
close to the active site exposed by a conformational
change at acidic pH. The following mechanism
may therefore be assumed. In porcine pepsinogen,
both cleavage sites may come close to the active
site and are similarly susceptible to the proteolysis
and therefore either bond is cleaved first to form
pepsin or the intermediate form. In monkey pep47
48
sinogen, the Leu-Ile bond may come closer to the
25
26
active site than the Asp-Phe bond and/or the
former may be more susceptible than the latter.
Thus the former is cleaved first, leading exclusively
to one-step activation. This hypothesis implies
that in pepsinogens two activation pathways are
always probable depending on the structure of
the activation segment and its location relative to
the active site.
Porcine pepsinogen has the same 2-residue
sequences as Japanese monkey pepsinogens, i.e.,
24
25
Asp-Phe, in the middle region of the activation
Vol. 93, No. 3, 1983
segment, but this bond was not cleaved in the
early period of activation. This may be due to
a slight difference in the length of the activation
segment. The intact activation segment of porcine
pepsinogen is composed of 44 residues and this
is 3 residues shorter than those of human and
monkey pepsinogens. The lack of 2 residues
was especially observed in the C-terminal region
16
17
of the porcine segment. Thus the Leu-Ile bond
24
25
rather than the Asp-Phe bond in the porcine segment may come close to the exposed active site
and be cleaved off. However, another possibility
cannot be excluded that the difference is partly
due to the difference in the susceptibility of the
peptide bonds themselves to the proteolysis. In
16
17
porcine pepsinogen the Leu-Ile bond may be more
24
25
susceptible than the Asp-Phe bond. On the other
25
26
hand, in monkey pepsinogen the Asp-Phe bond
17
18
may be more susceptible than the Leu-Ser bond
16
17
which corresponds to the Leu-Ile bond in porcine
pepsinogen.
We thank Drs. H. Umezawa and T. Aoyagi at the Institute of Microbial Chemistry, Tokyo, for the generous
supply of pepstatin.
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T. KAGEYAMA and K. TAKAHASHI