A serine endopeptidase from cucumber leaves is inhibited by L-arginine, guanidino compounds and divalent cations

Article abstract of DOI:10.1016/S0031-9422(01)00309-0

An endopeptidase was purified and characterized from green leaves of cucumber (Cucumis sativus L. suyo). The purified enzyme, a basic amino acid-specific endopeptidase with a pI of 5.0, was a monomeric protein of 80 kDa whose pH optimum was 9.5. Inhibitor analysis suggested that it was a serine endopeptidase and contained sulfhydryl groups essential for catalytic activity. Analysis of internal amino acid sequences of the endopeptidase showed no significant similarity to other proteins. Its activity was inhibited by L-Arg and guanidino compounds having high hydrophobicity, as well as divalent cations such as Mg²⁺ and Ca²⁺. The K_i values of L-Arg and Mg²⁺, which are also likely in vivo inhibitors, were 3.5 and 10 mM, respectively. Inhibition by L-Arg and Mg²⁺ was additive, and more than 70% of the activity was reversibly inhibited under their physiologically significant concentrations. These results suggest that the enzyme is possibly regulated by L-Arg and/or guanidino compounds, and by divalent cations in vivo.

Full text of DOI:10.1016/S0031-9422(01)00309-0

Phytochemistry 58 (2001) 677–682
www.elsevier.com/locate/phytochem
A serine endopeptidase from cucumber leaves is inhibited by
l-arginine, guanidino compounds and divalent cations
Yasuo Yamauchia,*, Toshio Sugimotob, Kuni Sueyoshib,1,
Yoshikiyo Ojib, Kiyoshi Tanakaa
aLaboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan
bDepartment of Biological and Environmental Sciences, Faculty of Agriculture, Kobe University, Rokkodai 1-1, Nada-ku, Kobe 657-8501, Japan
Received 15 February 2001; received in revised form 5 June 2001
Abstract
An endopeptidase was purified and characterized from green leaves of cucumber (Cucumis sativus L. suyo). The purified enzyme,
a basic amino acid-specific endopeptidase with a pI of 5.0, was a monomeric protein of 80 kDa whose pH optimum was 9.5. Inhi-
bitor analysis suggested that it was a serine endopeptidase and contained sulfhydryl groups essential for catalytic activity. Analysis
of internal amino acid sequences of the endopeptidase showed no significant similarity to other proteins. Its activity was inhibited
by l-Arg and guanidino compounds having high hydrophobicity, as well as divalent cations such as Mg2+ and Ca2+. The Ki values
of l-Arg and Mg2+, which are also likely in vivo inhibitors, were 3.5 and 10 mM, respectively. Inhibition by l-Arg and Mg2+ was
additive, and more than 70% of the activity was reversibly inhibited under their physiologically significant concentrations. These
results suggest that the enzyme is possibly regulated by l-Arg and/or guanidino compounds, and by divalent cations in vivo. # 2001
Elsevier Science Ltd. All rights reserved.
Keywords: Cucumis sativus; Cucurbitaceae; Serine endopeptidase; Trypsin-like; Inhibition; Arginine; Guanidino compound; Magnesium
1. Introduction
To overcome this problem, we have developed both
polyacrylamide gel electrophoretic and activity staining
methods to separate and detect low proteolytic activities,
as well as a method for detection of measurable endo-
peptidase activities using artificial substrates (Yamauchi
et al., 1995). These techniques were used to detect two
major endopeptidases that degraded benzoyl (Bz)-Arg-
naphthylamide (NA) and carbobenzoxy (CBZ)-Leu-Leu-
Glu-NA, respectively (Yamauchi et al., 1996). This
report describes the purification and characterization of
the Bz-Arg-NA degrading endopeptidase (CEP 5.0). The
enzyme was a basic amino acid-specific endopeptidase
that is plausibly regulated by l-Arg and/or guanidino
compounds and divalent cations whose cellular con-
centrations may change with environmental conditions.
In all organisms, protein turnover is a highly regulated,
selective process. It is well known that the mammalian cell
contains highly regulated protein degradation systems
such as ubiquitin-proteasome in the cytosol (Hershko
and Ciechanover, 1992) and the Ca2+, calpain, and cal-
pastatin systems (Sorimachi and Suzuki, 1998). Many
reports also imply the existence of highly regulated pro-
teolytic systems in plants, but only a few have described the
role played by plant endopeptidases in regulation of pro-
tein turnover (Vierstra, 1993; von Kampen et al., 1996).
It is difficult to investigate endopeptidases since they are
both present at low levels and occur in multiple isoforms.
Abbreviations: Bz, benzoyl; CBZ, carbobenzoxy; CEP, cucumber
endopeptidase; 2-ME, 2-mercaptoethanol; PB, phosphate buffer; NA,
naphthylamide
2. Results and discussion
* Corresponding author. Tel.: +81-857-31-6702; fax: +81-857-31-
6702.
E-mail address: yamauchi@muses.tottori-u.ac.jp (Y. Yamauchi).
The Bz-Arg-NA degrading endopeptidase was pur-
ified to apparent homogenets by successive chromato-
graphy on DEAE-Toyopearl, hydroxyapatite, phenyl-
1
Present address: Department of Applied Biological Chemistry,
Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00309-0

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Y. Yamauchi et al. / Phytochemistry 58 (2001) 677–682
sepharose, Arg-sepharose, and mono
Q
columns
The substrate specificity of CEP 5.0 was determined
to be trypsin-like by using various artificial substrates.
The enzyme cleaved artificial substrates such as Bz-Arg-
NA and Bz-Lys-p-nitroanilide, but did not cleave those
diagnostic for aminopeptidase, caspase, cathepsin D,
chymotrypsin, elastase, glutamyl endopeptidase and
subtilisin (data not shown). Plant serine endopeptidases
of 80–82 kDa whose pH optimum are at alkaline pH,
have been isolated from ragweed, Euphorbia supina and
bamboo (Bagarozzi et al., 1996; Arima et al., 2000a,b);
however, their substrate specificities are quite different
from CEP 5.0, i.e. they have chymotyrpsin-like or sub-
tilisin-like activities. CEP 5.0 degraded gelatin, but did
not degrade azocasein, carboxymethyl-BSA, acid-dena-
tured haemoglobin and Rubisco (data not shown).
Since amino acid sequencing of the N-terminus of CEP
5.0 showed that it was blocked, internal amino acid
sequences were analyzed after CNBr cleavage treatment
of purified CEP 5.0 and separation of the cleaved peptides
by SDS-PAGE. The N-terminal amino acids sequences of
a 25 and a 20 kDa polypeptide were ‘‘FSLDLEAQE’’
and ‘‘PTGPQAQQE’’, respectively. Homology search of
the Swissplot and translated EMBL databases revealed
(Table 1). The endopeptidase was named CEP 5.0
because the purified enzyme migrated as a single band
(pI=5.0) when submitted to nondenaturing isoelectric
focusing (Fig. 1A). The molecular mass of CEP 5.0 was
80 kDa as estimated by both SDS-PAGE and gel filtra-
tion (Fig. 1B and C), suggesting that CEP 5.0 was an 80
kDa monomeric protein. The enzyme, with an optimum
pH of around 9.5, showed half-maximal activities at pH7.4
and 10.5. CEP 5.0 was most active at 50 ꢀC and was com-
pletely denatured when incubated at 70 ꢀC (data not
shown).
As shown in Table 2, diisopropyl fluorophosphate
and phenylmethylsulfonyl fluoride reduced the activity
of CEP 5.0 as did leupeptin, suggesting that CEP 5.0 is a
serine endopeptidase. Enzyme inhibitors such as p-
chloromercuribenzoic acid and N-ethylmaleimide also
inhibited CEP 5.0. This suggests that CEP 5.0 contained
sulfhydryl groups essential to its catalytic activity.
Table 2
Effects of inhibitors on CEP 5.0 activity
Inhibitor
Concentration
Relative
activity (%)
Control
Diisopropyl fluorophosphate
–
100a
6.8
1 mM
10 mM
1 mM
10 mM
0.5 mM
2.5 mM
1 mM
10 mM
10 mM
10 mM
0.1%
0
Phenylmethylsulfonyl fluoride
p-Chloromercuri benzoic acid
N-Ethylmaleimide
79.3
22.4
42.9
25.3
21.3
11.6
0.9
Fig. 1. Estimation of pI and molecular mass of CEP 5.0: (A) 1 mg of
purified CEP 5.0 was subjected to isoelectric focusing on a 7.5% (w/v)
acrylamide, 2% SERVALYT (pH 4.0–6.0) gel; (B) 1 mg of purified
CEP 5.0 was subjected to SDS-PAGE on a 13% (w/v) acrylamide gel
under reducing conditions. The molecular mass of protein markers are
given in the lane M. (C) Plot of the Rf values against log molecular
mass for CEP 5.0 (*) and marker proteins (&) on Superose 12 col-
umn. Marker proteins were ferritin (450 kDa), catalase (240 kDa),
bovine serum albumin (67 kDa), egg albumin (45 kDa), and chymo-
trypsinogen A (25 kDa).
Leupeptin
Pepstatin A
78.4
127
0
Soybean trypsin inhibitor
SDS
0.1%
a
Activity in the absence of inhibitor is defined as 100% (0.28 units
of Bz-Arg-NA cleaving activity).
Table 1
Summary of purification of CEP 5.0
Step
Total protein
(mg)
Total activity
(Units)a
Specific activity
(Units/mg)
Purification-
fold
Yield
(%)
Crude extract
45–55%
4430
653.6
89 090
82 600
22.15
130
1.0
5.87
100
84.2
Ammonium sulfate
DEAE-Toyopearl
Hydroxyapatite
Phenyl-sepharose
Arg-sepharose
MonoQ
20.16
7.26
1.46
25 980
21 500
10 600
12 880
1627
1289
2960
58.2
133.7
328.5
634.6
2998
26.5
21.9
10.8
13.1
7274
14 050
66 400
0.917
0.0245
1.66
a
Bz-Arg-NA was used as a substrate, and one unit was defined as the formation of 1 nmol of naphthylamine per minute.

Y. Yamauchi et al. / Phytochemistry 58 (2001) 677–682
679
no significant similarity of these sequences to other
proteins.
(Fig. 2C). Methylester and ethylester derivatives became
stronger inhibitors, and l-Arg modified by naphthyla-
mine and nitroaniline almost completely inhibited CEP
5.0 at much lower range. Ng-methyl-l-Arg which is a
guanidino group-methylated l-Arg-derivative, showed an
increase of inhibition. These results indicate that strong
inhibition is due to hydrophobicity of l-Arg-derivatives,
and that Bz-Arg-NA showed substrate-inhibition because
it had hydrophobic bases on both the amino- and car-
boxyl-groups of l-Arg. Inhibition by l-Arg-derivatives
Although substrate inhibition at low substrate levels
(Fig. 2A) prevented the determination of the Km for
Bz-Arg-NA, the Km for Bz-Lys-p-nitroanilide was 50
mM. Thus, Bz-Lys-p-nitroanilide was used as the sub-
strate in subsequent kinetic experiments. To determine
what portion of Bz-Arg-NA inhibited CEP 5.0, we first
examined the effects of amino acids on CEP 5.0 activity.
Only l-Arg significantly inhibited CEP 5.0 (Fig. 2B),
whereas other amino acids, Ala, Asn, Glu, Gln, His, Ile,
Lys, Met, l-ornithine, Phe, Pro, Ser, Val, Tyr showed lit-
tle or no inhibitory effects. The Ki value of l-Arg was
3.5 mM and about 90% of activity was inhibited in the
presence of 20 mM l-Arg (Fig. 2B).
The increased inhibition with Bz-Arg-NA compared to
l-Arg (Fig. 2A and B) suggests that modification of l-
Arg caused the increased inhibition. The effect of mod-
ification of l-Arg on inhibitory activity using various l-
Arg-derivatives was further examined (Fig. 3A and
Table 3). Na-amino group modified l-Arg-derivatives
such as acetyl-l-Arg slightly changed its inhibitory
activity, compared with the large inhibition observed
with Bz-l-Arg (Fig. 2B). Modification of the carboxyl
group of l-Arg increased inhibitory activity drastically
Fig. 3. Structures of l-Arg-derivatives and guanidino compounds
using in this study: (A) l-Arg-derivatives modified by various chemical
groups; (B) l-Arg and amino acids whose structures are similar to l-
Arg; (C) guanidino compounds observed in plants. Asterisk shows
substance observed in plants.
Table 3
Summary of kinetic analysis of l-Arg-derivatives against CEP 5.0
activity
Fig. 2. Inhibition of CEP 5.0 by various Arg-derivatives and divalent
cations: (A) CEP 5.0 was incubated with various concentrations of Bz-
Lys-p-nitroanilide (&) or Bz-Arg-NA (&) as the substrates; (B) CEP
5.0 activity was measured with various concentrations of l-Arg (*),
l-Lys (x) or Na-modified l-Arg-derivatives; acetyl-l-Arg (~) or Bz-l-
Arg (&). (C) CEP 5.0 activity was measured with various concentra-
tions of C-modified l-Arg-derivatives; l-arginine methylester (~), l-
arginine ethylester (&), l-Arg-p-nitroanilide (*) or l-Arg-NA (*). (D)
CEP 5.0 activity was measured with various concentrations of Mg2+ (x)
or Ca2+ (*). Activity in the absence of effector was defined as 100%.
The values represent the average of at least three independent measure-
ments using 0.27 units of Bz-Lys-p-nitroanilide cleaving activity of CEP
5.0 and the range of the experimental error was within Æ3%.
Site modified l-Arg-derivatives
Intact
Na-modified Acetyl-l-Arg
Bz-l-Arg
Ki (mM) Inhibitory fashion
l-Arg
3.5
2.5
Competitive
Competitive
Competitive
Competitive
0.39
0.19
C-modified
l-Argininamide
l-Arginine-methylester 94 Â 10À3 Competitive
l-Arginine-ethylester
l-Arg-p-nitroanilide
l-Arg-NA
66 Â 10À3 Competitive
58 Â 10À3 Non-competitive
14 Â 10À3 Non-competitive
Ng-modified Ng-Methyl-l-Arg
1.5
Competitive
0.27 units of Bz-Lys-p-nitroanilide cleaving activity was used.

680
Y. Yamauchi et al. / Phytochemistry 58 (2001) 677–682
was competitive except for non-competitive inhibition
by l-Arg-p-nitroanilide and l-Arg-NA (Fig. 4).
Table 4
Summary of kinetic analysis of guanidino compounds detected in
plants against CEP 5.0 activity
Neither l-citrulline, which lacks a positively charged
guanidino group (structure is referred to in Fig. 3B), nor
l-Lys and l-ornithine that have a positive charge at
physiological pH, inhibited the reaction (data not
shown). l-Argininosuccinic acid, in spite of having
positively charged guanidino group, also did not inhibit
activity, indicating steric hindrance by the large succinic
group. These results suggest that an intact positively
charged guanidino group at physiological pH is essen-
tial for inhibition.
Guanidino compounds
Ki (mM)
Inhibitory fashion
a
Guanidinoacetic acid
–
>20
–
Guanidinopropionic acid
Guanidinobutyric acid
Guanidinobutylamide (agmatine)
Homoarginine
Competitive
Competitive
Competitive
Competitive
2.8
0.5
30 Â 10À3
0.27 units of Bz-Lys-p-nitroanilide cleaving activity was used.
a
Not inhibited.
We examined whether other guanidino compounds
observed in plant tissues also inhibit CEP 5.0 (structures
are referred to Fig. 3B and C, Table 4). Guanidino butylic
acid showed almost equal inhibition to l-Arg; however,
guanidino propionic acid showed less inhibition and gua-
nidino acetic acid showed no inhibitory activity, suggest-
ing that shortening of the hydrocarbon chains of
guanidino compounds decreased inhibitory activity.
Homoarginine, which has a longer hydrocarbon chain
than l-Arg, and guanidinobutylamine (agmatine) showed
strong inhibition at much lower range than l-Arg.
Although these highly hydrophobic guanidino com-
pounds only occur at low levels (Kato et al., 1986), it is
possible that they and other unidentified guanidino
compounds might regulate CEP 5.0 activity in vivo.
Arg is thought to be an endogenous inhibitor of CEP
5.0 because plants can contain Arg up to 5 mM (Winter
et al., 1993). As Arg is the most economic utilizer of
nitrogen because of its high N/C ratio (4N:6C), plants
usually use Arg for storage and transport of N. Gen-
erally Arg contents in plants are affected by nutrient
conditions and aging. For example, Arg content in
leaves of young tulips cultivated in a medium containing
nitrogen was over 70 times higher than those in leaves
after flowering and in nitrogen starved conditions
(Ohyama et al., 1985). Therefore, CEP 5.0 may be regu-
lated in response to nutrient and developmental changes
in Arg content. A few endopeptidases inhibited by l-Arg
have been reported in animals; however, the inhibition
was less effective than the case for CEP 5.0 (Hatton, 1973;
Polakoski and McRorie, 1973), and no plant endopepti-
dase regulated by Arg has been reported.
CEP 5.0 was also competitively inhibited by divalent
cations such as Mg2+ and Ca2+ (Figs. 2D and 4B). This
indicates that Mg2+ and Ca2+ interferes with binding
of the substrate, either due to binding of themselves to
the catalytic site or to a closely related site. The Ki value
of inhibition by divalent cations was about 10 mM.
Divalent cations such as Ca2+ and Mg2+ are well
known regulators of plant endopeptidases (Liu and
Jagendorf, 1986; Bushnell et al., 1993; Anastassiou and
Argyroudi-Akoyunoglou, 1995), and may play impor-
tant roles in protein degradation of plants. The most
likely divalent cation for regulation of CEP 5.0 in vivo is
Mg2+ because it is the most abundant soluble divalent
cation while intracellular free Ca2+ is usually kept to low
levels (Kreimer et al., 1988). The concentration of Mg2+ is
assumed to be 13–17 mM in the vacuole and 2–10 mM in
cytosol and chloroplasts (Leigh and Wyn Jones, 1986;
Stelzer et al., 1990; Marschner, 1995). These concentra-
tions are high enough to inhibit CEP 5.0.
Moreover, we examined the inhibition of CEP 5.0 in
the presence of various physiological concentration of
l-Arg and Mg2+, which are the most likely inhibitors in
vivo (Fig. 5). Inhibition by l-Arg and Mg2+ was addi-
tive and reversible (data not shown), and 5 mM of l-
Arg and Mg2+ inhibited the enzyme activity more than
70% compared to the control activity. This suggests the
plausible regulation of CEP 5.0 by physiological con-
centration of l-Arg and Mg2+ in vivo.
In conclusion, CEP 5.0 is possibly regulated by l-Arg
and/or guanidino compounds and Mg2+ whose cellular
concentrations may change with environmental condi-
tions. To clarify the physiological role of CEP 5.0, we
will investigate in future the native substrate and locali-
zation of CEP 5.0.
Fig. 4. Kinetic analysis of the inhibition of CEP 5.0 on Hanes–Woolf
plots: (A) CEP 5.0 was incubated without (*) or with 5 mM (&) or 10
mM (Á) l-Arg-NA; (B) CEP 5.0 was incubated without (*) or with l-
Arg-derivatives. Symbols are 10 mM Bz-l-Arg (+), 0.2 mM Ng-
methyl-l-Arg (*), 4 mM l-Arg (x), 0.1 mM l-arginine methylester
(&), or 5 mM Mg2+ (~), respectively. The values represent the aver-
age of at least three independent measurements using 0.27 units of Bz-
Lys-p-nitroanilide cleaving activity of CEP 5.0 and the range of the
experimental error was within Æ3%.

Y. Yamauchi et al. / Phytochemistry 58 (2001) 677–682
681
layers of cheesecloth. After centrifugation at 20,000 g for
20 min, the supernatant was fractionated by the addition
of solid ammonium sulfate. The fraction precipitating
between 45 and 55% ammonium sulfate saturation was
dissolved in a minimum volume of 25 mM K-PB, pH 7.0,
containing 1 mM 2-ME (buffer A) and dialyzed against
buffer A. The dialysate was applied to a DEAE-Toyopearl
column (4Â20 cm, Tosoh Corp., Japan) equilibrated with
buffer A, and a linear gradient of 0–0.2 M NaCl (total 1.2
l) was applied to elute the enzymes. Active fractions of
CEP 5.0 after DEAE-Toyopearl chromatography were
combined and dialyzed against 10 mM Na-PB, pH 6.8,
containing 1 mM 2-ME. The dialysate was applied to a
hydroxyapatite column (2Â15 cm, Bio-Rad, CA, USA)
and eluted by a linear gradient of 0–0.2 M Na-PB (total
200 ml). After active fractions were combined, an equal
volume of 60% saturated ammonium sulfate in 50 mM K-
PB, pH 7.0, containing 1 mM 2-ME was added. This
solution was applied to a Phenyl-Sepharose column
(1.5Â8 cm, Amersham Pharmacia Biotech, Sweden) equi-
librated with 30% ammonium sulfate saturated in 50 mM
K-PB, pH 7.0, containing 1 mM 2-ME and the proteins
were eluted by a linear gradient of 30–0% ammonium sul-
fate (total 100 ml). Active fractions were concentrated by a
DEAE-mini column (1 ml), then applied to an Arg-
Sepharose (1.5Â8 cm, Amersham Pharmacia Biotech,
Sweden) column equilibrated with buffer A containing
0.1 mM EDTA. A linear gradient of 0–0.1 M NaCl was
applied to elute the enzyme (total 100 ml). The active
fractions were dialyzed against buffer A, then applied to
a Mono Q column (0.32Â3 cm, SMART system,
Amersham Pharmacia Biotech, Sweden). The column
was eluted with a linear gradient of 0–0.3 M NaCl in
buffer A (total 8 ml). All (purificatiꢀon) steps were per-
formed at temperatures from 0 to 4 C.
Fig. 5. Inhibition of CEP 5.0 by l-Arg and Mg2+ is additive. CEP 5.0
(0.27 units of Bz-Lys-p-nitroanilide cleaving activity) was incubated
with various combination of l-Arg and Mg2+ concentration. Activity
in the absence both of l-Arg and Mg2+ was defined as 100%.
3. Experimental
3.1. Materials
All of the Arg-derivatives and Fast Garnet GBC were
purchased from Sigma Co. Ltd. (St. Louis, MO, USA).
Leupeptin and pepstatin A were purchased from Peptide
Institute, Inc. (Osaka, Japan). All other reagents were
purchased from Wako Pure Chemical Co. Ltd. (Osaka,
Japan). Cucumber seeds (Cucumis sativus L. suyo) pur-
chased from Takii Seed Co. Ltd. (Kyoto, Japan), were
ꢀ
germinated in the dark at 25 C for 6 days then trans-
ferred to a greenhouse. Harvested leaves were frozen in
liquid N2 and stored below À20 ꢀC until use.
3.2. Enzyme activities
3.4. Characterization of CEP 5.0
To monitor benzoyl (Bz)-Arg-naphthylamide (NA)
degrading activity, assays using 60 mM of Bz-Arg-NA as
a substrate by the method described previously were
employed (Yamauchi et al., 1996). For kinetic analysis
of the endopeptidase, 160 mM of Bz-Lys-p-nitroanilide
was used as a substrate in 50 mM Hepes-KOH, pH 8.0,
to a final volume of 1 ml. After incubation at 37 ꢀC, the
reaction was stopped by addition of 0.5 ml of 30% (v/v)
acetic acid and absorbance at 410 nm measured. One
unit was defined as the formation of 1 nmol of nitroa-
niline or naphthylamine per minute.
To investigate the effect of inhibitors, CEP 5.0 was pre-
incubated with each inhibitor inꢀthe reaction mixture
without substrate for 30 min at 4 C. The substrate was
then added to the mixture and residual activity was mea-
sured. Native molecular weights were estimated on the
basis of comparison of the retention time of marker pro-
teins on a Superose 12 column (0.32Â30 cm, SMART
system, Amersham Pharmacia Biotech, Sweden) equili-
brated with 25 mM K-PB, pH 7.0, containing 0.1 M NaCl
and 1 mM 2-ME. In the experiment on the effect of l-Arg-
derivatives on CEP 5.0, l-Arg-derivatives were used as
inhibitors because CEP 5.0 could not cleave them even
though their structure resemble the Bz-Arg-NA substrate.
3.3. Purification of cucumber endopeptidase with a pI of
5.0 (CEP 5.0)
Leaves (300 g) were extracted with 5 volumes of 50
mM potassium phosphate buffer (K-PB), pH 7.0, con-
taining 2 mM 2-mercaptoethanol (2-ME) and 0.1 mM
EDTA. Debris was removed by filtration using two
3.5. Protein analysis
Isoelectric focusing was performed with a 7.5% (w/v)
polyacrylamide gel containing SERVALYT of pH

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Y. Yamauchi et al. / Phytochemistry 58 (2001) 677–682
range 4.0–6.0 (Serva Electrophoresis GmbH, Germany)
under non-denaturing conditions. Cathode and anode
buffers were 40 mM Glu and 0.2 M His, respectively.
SDS-PAGE and Tricine/SDS-PAGE were performed by
the method of Laemmli (1970) and Schagger and von
Jagow (1987), respectively. Gels were stained for proteins
with Coomassie Brilliant Blue R-250. Protein concentra-
tion was determined by the method of Bradford with
bovine serum albumin as the standard (Bradford, 1976).
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103, 585–591.
Hatton, M.W.C., 1973. Studies on the coagulant enzyme from Agkis-
trodon rhodostoma venom. Biochem. J. 131, 799–807.
Hershko, A., Ciechanover, A., 1992. The ubiquitin system for protein
degradation. Ann. Rev. Biochem. 61, 761–807.
Kato, T., Kondo, T., Mizuno, K., 1986. Occurrence of guanidino
compounds in several plants. Soil Sci. Plant Nutr. 32, 487–491.
Kreimer, G., Melkonian, M., Holtum, J.A.M., Latzko, E., 1988.
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3.6. Sequencing of internal peptides of CEP 5.0
CEP 5.0 (2 mg) was dissolved in 1 ml of 70% formic
acid containing 1 mg of CNBr, and allowed to stand
overnight in the dark. After evaporation using a SpeedVac
concentrator (model A160, SAVANT Instruments Inc.,
NY, USA), the cleaved peptides were dissolved in 50 ml of
SDS sample buffer and separated by Tricine/SDS-PAGE
using a 15% polyacrylamide gel. After electroblotting to a
poly (vinylidene difluoride) membrane, peptides were
stained by CBB R-250 and portions of major bands were
excised and sequenced by an automated pulsed liquid
protein sequencer (model 491, Applied Biosystems,
Foster City, CA, USA).
Marschner, H., 1995. Mineral Nutrition of Higher Plants. Academic
Press, San Diego, CA, pp. 277–285.
Ohyama, T., Ikarashi, T., Baba, A., 1985. Nitrogen accumulation in
the roots of tulip plants (Tulipa gesneriana). Soil Sci. Plant Nutr. 31,
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