Journal of Biotechnology 100 (2003) 221 /229
www.elsevier.com/locate/jbiotec
Isolation of H-translocating ATPase in tonoplast of
Tradescantia virginiana L. leaf cells
Mikako Saito a, Hideaki Matsuoka a,*, Kunihiro Kasamo b
a
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei,
Tokyo 184-8588, Japan
b
Research Institute for Bioresources, Okayama University, 2-20-1, Chuo, Kurashiki, Okayama 710-0046, Japan
Received 24 April 2002; received in revised form 11 July 2002; accepted 22 July 2002
Abstract
The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate
(DOC) and n -octyl-b-D-glucoside (n -OG). Three major polypeptides (68, 60, 16 kDa) and several other minor
components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H pump
activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine /
fluorescence quenching was observed in the presence of ATP /Mg2 , indicating the H pumping. The H pump
activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO3, an inhibitor specific to tonoplasttype (V-type) H -ATPase.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Tonoplast; H -ATPase; Tradescantia virginiana
Abbreviations: BSA, bovine serum albumin; DCCD, N ,N ?dicyclohexylcarbodiimide; DES, diethylstilbestrol; DOC,
deoxycholate; DTT, dithiothreitol; EDTA, ethylenediamineN ,N ,N ?,N ?-tetraacetic acid; EGTA, ethylene glycol-bis (bamino ethyl ether) N ,N ,N ?,N ?-tetraacetic acid; MES, 2-(N morpholino) ethanesulfonic acid; MOPS, 3-(N -morpholino)
propanesulfonic acid; n -OG, n -octyl-b-D-glucoside; PMSF,
phenylmethylsulfonyl fluoride; PVP, polyvinylpyrrolidone;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis; TCA, trichloroacetic acid.
* Corresponding author. Tel.: /81-42-388-7029; fax: /8142-387-1503
E-mail address: [email protected] (H. Matsuoka).
1. Introduction
Experimental evidences of self-defense mechanisms, which plants show in response to various
stresses from their environment have recently been
accumulated. Some cases of those mechanisms
have already been elucidated at molecular level
(Farmer and Ryan, 1992). Since several years ago,
the authors have investigated the response of a leaf
to CO2 stress; i.e. extremely high concentration
and non-physiological condition of CO2 gas (Mat-
0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 2 4 4 - 4
222
M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
suoka et al., 1986). The exposure of CO2 for a
short period caused a marked response of intracellular potential change in plant leaf cells. The
response was reproducible and seemed to reflect
the physiological condition of respective sample
leaves. Therefore, its response pattern was expected to be a useful indicator of the healthy
condition of a leaf that had been exposed to more
toxic gases such as NOx, SOx. These suggested the
use of a plant leaf as a bioelectrochemical indicator of atmospheric pollution.
In order to analyze the molecular mechanism of
the electrochemical response of a plant leaf, the
authors prepared ion sensitive microelectrodes and
measured the dynamic responses of H , K , and
Cl concentrations, respectively (Saito et al.,
1993). The intracellular space of a plant cell was
composed of a large volume of vacuole and a
narrow space of cytosol. Thus the concentrations
of those three ions were measured both in the
cytosol and the vacuole, respectively. Contrary to
our expectation, the active transport of K from
the vacuole to the cytosol occurred at first earlier
than the H flow. According to a well understood
mechanism of the ion transport, the energy source
is the H -ATPase and, therefore, the H flow
driven by the H pump should occur before active
transport of any other ions.
To elucidate this unexpected phenomenon, it
was thought to be necessary to isolate the molecules supposedly involved in these ion transportations and to present their respective functions, for
example, by the reconstitution method. As the first
step, we intended to confirm the presence of H translocating pump in the tonoplast of Tradescantia virginiana leaf cells.
According to the former studies, H -translocating ATPase in the tonoplast of other plant species
is vacuolar (V) type, which is different from
chloroplasts or mitochondria (F) (Futai et al.,
1989) and plasma membrane (P) (Pedersen and
Carafoli, 1987) types in their subunit structure,
ATP synthesis activity and susceptibility to specific
inhibitor. In this study, H -translocating ATPase
is isolated from T. virginiana leaf cells and its
characteristics are investigated.
2. Materials and methods
2.1. Materials
Young plants of T. virginiana were purchased
from Nihon Kaki Co., Kawaguchi, Saitama, and
cultivated at 28 8C in a greenhouse.
2.2. Isolation of tonoplast
Approximately 700 g fresh weight of leaves of T.
virginiana were homogenized in a chilled mortar
with a pestle in 2800 ml of grinding buffer that
contained 0.25 M sorbitol, 5 mM EGTA, 1 mM
PMSF, 2.5 mM potassium metabisulfite, 1.5%
PVP and 50 mM MOPS/KOH (pH 7.6). The
homogenate was filtered through four layers of
gauze and the filtrate was centrifuged at 3600/g
for 10 min. The supernatant was further centrifuged at 150 000 /g for 20 min. The pellet was
suspended in 6 ml of a sucrose buffer solution
which consisted of 0.3 M sucrose, 1 mM EGTA, 2
mM DTT and 10 mM potassium phosphate buffer
(pH 7.8). Three millilitre of a MOPS buffer
solution containing 0.25 M sorbitol, 1 mM
EGTA, 2 mM DTT and 5 mM MOPS/KOH
(pH 7.3) was layered over this suspension. The
step gradients were centrifuged at 120 000 /g for
40 min. The interface was collected, diluted 5-fold
with the MOPS buffer and centrifuged at
150 000 /g for 20 min. The pellet thus obtained
was suspended in the MOPS buffer solution and
layered over a sucrose buffer solution again. The
centrifugation (120 000 /g for 40 min) was repeated and the interface was collected, diluted 5fold with the MOPS buffer solution and centrifuged at 150 000/g for 20 min. The resulting
pellet was resuspended in a suspending medium
containing 0.25 M sorbitol, 2 mM DTT, 5 mM
MES/Tris (pH 7.5) and 15% glycerol.
2.3. Solubilization of tonoplast ATPase
A two-step procedure using DOC and n -OG
was carried out to solubilize ATPase from the
tonoplast as described previously (Kasamo et al.,
1991). In brief, in the first step, a 10% stock
solution of DOC was added drop-wise to the
M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
223
tonoplast suspension in the solubilization buffer A
that contained 3 mM EDTA, 3 mM DTT and 30
mM Tris/MES (pH 7.5), with stirring on ice. The
final concentration of DOC was 0.1%, and the
concentration of protein was approximately 1 mg
ml 1. After 15 min stirring, the suspension was
centrifuged at 140 000/g for 1 h. Thus obtained
pellet contained the membrane fraction. In the
second step, solubilization buffer B that contained
1 mM EDTA, 1 mM DTT, 10 mM Tris/MES (pH
7.5) and 45% glycerol was added to the pellet to
give a final concentration of protein at approximately 2 mg ml1. A 300 mM stock solution of nOG was added to the pellet at a final concentration
of 40 mM. After 15 min stirring and centrifugation
at 140 000 /g for 1 h, most of the ATPase activity
was found in the supernatant.
homogenizer resulted in a clear suspension. This
detergent/protein /lipid mixture was applied to
the top of a column of PD-10 (Pharmacia,
Uppsala) equilibrated with a washing buffer that
contained 0.1 mM EDTA, 100 mM KCl and 10
mM MES/Tris (pH 7.2). The column was eluted
with the washing buffer at a flow rate of 1 ml
min1. The cloudy fractions eluted after the void
volume were pooled, diluted with the washing
buffer and centrifuged at 100 000 /g for 1 h. The
pellet was suspended in the washing buffer for the
assay of H -pumping.
2.4. Reconstitution of tonoplast H -ATPase into
liposomes
2.6. Assay for ATPase activity
Tonoplast H -ATPase from the plasma membrane was reconstituted according to the method
described by Perlin et al. (1984) and Kasamo et al.
(1991) with slight modification. An important
point is the use of asolectin because asolectin was
formerly found effective in the reconstitution of
tonoplast ATPase from mung bean (Kasamo et
al., 1991) as well as the reconstitution of bacteriorhodopsin (Racker et al., 1979), bacterial
NADH-dehydrogenase (Baron and Thompson,
1975) and H -ATPase from Neurospora crassa
(Perlin et al., 1984). Asolectin was purified and
stored in a mixture of chloroform and methanol at
/20 8C, as described by Kagawa and Racker
(1971). At first, the chloroform /methanol mixture
was removed from asolectin under a gentle stream
of N2 to form lipid film on the inner surface of the
glass tube. Reconstitution buffer containing 1 mM
EDTA, 100 mM KCl and 10 mM MES/Tris (pH
7.2) was added to the tube and the suspension was
sonicated in a bath-type sonicator. Fifty microgram of solubilized ATPase was diluted with
reconstitution buffer to 1.2 ml, into which purified
asolectin suspension obtained above was added.
Ten percent DOC was added to this cloudy
suspension to make a final concentration at
0.6%. Vigorous homogenization with a Teflon
ATPase activity assay was carried out by 30 min
incubation of 500 ml reaction mixture at 38 8C.
The reaction mixture contained 3 mM Tris /ATP,
3 mM MgSO4, 30 mM MES/Tris (pH 7.5) and 50
mM KCl. In order to characterize the ATPase,
either of the following inhibitors was added to the
reaction mixture: 2 mM gramicidin D, 100 mM
DCCD, 100 mM DES, 100 mM sodium azide, 100
mg ml 1 oligomycin, 2 mM Na3VO4, or 2.5 M
KNO3. The reaction was initiated by adding a
sample containing 10 mg protein to the reaction
mixture. After 30 min, the reaction was stopped by
the addition of 100 ml 50% TCA and liberated Pi
was measured as follows. An aliquot (500 ml) of
the reaction mixture was pipetted into a test tube
containing 500 ml 2% sodium molybdate, 500 ml 1.5
N H2SO4 and 2 ml iso -butanol, then mixed
vigorously for 10 s. After standing, the mixture
was clearly separated into an iso -butanol layer and
a water layer. Aliquote (1 ml) of the iso -butanol
layer were pipetted into other test tubes. One
millilitre of 0.5% ascorbic acid and 500 ml ethanol
were added successively to each of them. After
vigorous mixing for 10 s, the test tubes were
incubated at 38 8C for 30 min. The absorbance
measured at 720 nm with a spectrophotometer
(Model 228, HITACHI, Tokyo) corresponded to
Pi concentration.
2.5. Protein determination
Protein content was determined by the method
of Bradford (1976) using BSA as a standard.
224
M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
2.7. Estimation of H -translocating activity
The H -translocating activity was estimated by
following the pH change in the tonoplast or
liposomal vesicles. The decrease in the internal
pH of vesicle was determined from the quenching
of quinacrine/fluorescence (Kasamo, 1986).
Membrane vesicles (100 mg protein) were added
to a fluorescence assay solution consisting of 0.25
M sucrose, 50 mM KCl, 10 mM Tris/MES (pH
7.0) and 2 mM quinacrine at a final volume of 2
ml. After adding 120 ml of 50 mM ATP /MgSO4 to
the membrane vesicle suspension, the decrease in
the fluorescence at 500 nm was monitored using a
fluorescence spectrophotometer (Model FP-777,
JASCO, Tokyo) being excited at 425 nm. In order
to characterize the H -translocating ATPase,
either of the following inhibitors was added to
the fluorescence assay solution: 2 mM gramicidin
D, 2 mM Na3VO4 or 2.5 M KNO3.
2.8. Estimation of the type of H -translocating
ATPase
As described above, V-type H -translocating
ATPase is different from F- and P-types in its
susceptibility to specific inhibitor. V-type is inhibited by high concentration of KNO3, while Fand P-types are not. In contrast, F- and P-types
are inhibited by oligomycin and Na3VO4, while Vtype is not.
Therefore, either KNO3 or oligomycin, Na3VO4
was added to the reaction mixture and its effects
on the ATPase activity and H -translocating
activity were examined.
3. Results
3.1. ATPase activity of isolated tonoplast and
effects of inhibitors
Isolated tonoplast showed ATPase activity;
ATP hydrolysing activity of 14.2 mmol Pi per mg
protein h1 as shown in Table 1. The effects of
various inhibitors on the ATPase activity were
investigated. As shown in Fig. 1, DES and DCCD
inhibited it markedly around 0.1 mM, while NaN3
and oligomycin showed no inhibitory effect up to
100 mM. These results were common to every type
of ATPase as reported elsewhere. On the other
hand, the present sample was markedly inhibited
by KNO3 around 100 mM, while not by Na3VO4.
3.2. H -translocating activity of isolated tonoplast
vesicles
The capacity of ATP-driven H -transport
across the tonoplast was measured by the quenching of quinacrine/fluorescence. As shown in Fig.
2, the addition of ATP /Mg2 to the tonoplast
suspension induced marked quenching, indicating
net pumping of protons into the tonoplast vesicles.
The protons thus driven by the ATP-dependent
ATPase were immediately leaked out of the
vesicles by the addition of gramicidin D. As
predicted from the results of Fig. 1, this proton
pumping was inhibited by KNO3 but not by
Na3VO4.
2.9. SDS-PAGE
3.3. ATPase activity of solubilized H -ATPase
SDS polyacryamide gel electrophoresis was
carried out according to the method of Laemmli
(1970), using a 1.0 mm-thick slab gel consisting of
12.5% running gel and 4.5% stacking gel. Proteins
were suspended in a solution containing 2% SDS,
1% b-mercaptoethanol and 0.12 M Tris (pH 6.8),
and heated in a boiling water bath for 2 /3 min.
The running buffer consisted of 0.2 M glycine, 0.25
M Tris and 0.1% SDS. The gels were stained and
destained by the method of Fairbanks et al. (1971).
ATPase was solubilized from the tonoplast by
the two-step solubilization procedure using DOC
and n -OG. The specific activity of the ATPase
increased about 6.5-fold from 14.2 to 92.3 mmol Pi
per mg protein h1 as shown in Table 1. This
activity further increased by the addition of 0.03%
asolectin. The effects of various inhibitors on
isolated ATPase were the same as those observed
with tonoplast as summarized in Table 1.
M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
225
Table 1
Effects of inhibitors on tonoplast, solubilized ATPase and reconstituted ATPase
Inhibitor
Tonoplast (mmol Pi
per mg protein h 1)
Solubilized ATPase
(mmol Pi per
mg protein h 1)
Solubilized ATPase
with asolectin (mmol Pi
per mg protein h 1)
Reconstituted ATPase
(mmol Pi per
mg protein h 1)
None
DCCD (100 mM)
DES (100 mM)
NaN3 (100 mM)
Oligomycin (10 mg ml 1)
KNO3 (100 mM)
Na3VO4 (100 mM)
14.2
6.4
5.8
11.5
12.1
7.5
13.3
92.3
6.1
5.7
75.7
76.6
8.5
85.8
173.0
12.3
11.1
140.0
145.0
15.6
161.0
170.0
20.7
19.7
136.0
139.0
15.5
156.0
(100)
(45)
(41)
(81)
(85)
(53)
(94)
(100)
(7)
(6)
(82)
(83)
(9)
(93)
(100)
(7)
(6)
(81)
(84)
(9)
(93)
(100)
(12)
(12)
(80)
(82)
(9)
(92)
ATPase activity was determined in the presence of 3 mM ATP /MgSO4, 50 mM KCl and 30 mM MES/Tris (pH 7.5). Numbers in
parentheses indicate percentage of the control.
3.4. ATPase activity of reconstituted H -ATPase
H -ATPase was reconstituted into liposomes.
Phospholipid to protein ratios were varied. From
the results of Fig. 3, the optimal ratio for the
reconstitution of H -ATPase was found to be
200. In subsequent experiments, proteoliposomes
were reconstituted using a protein /phospholipid
mixture at a weight ratio of 1:200.
The specific activity of ATPase in the reconstituted proteoliposomes was much higher than that
of the ATPase in tonoplast vesicles as listed in
Table 1. As shown in Fig. 4, the activity of ATPase
in reconstituted proteoliposomes was inhibited by
KNO3 much more markedly than that in tonoplast. The optimum pH for ATPase in the
proteoliposome was 7.5 (Fig. 5).
3.5. Proton pumping activity of reconstituted H ATPase
The ability to drive protons into reconstituted
proteoliposomes was examined. Intraliposomal
acidification was monitored by the quenching of
quinacrine /fluorescence. Fig. 6 shows the quenching caused by the addition of ATP /Mg2 to the
reconstituted proteoliposomes which indicates the
net pumping of protons into the proteoliposomes
by the tonoplast ATPase. The quenching was
observed samely in the presence of Na3VO4. The
ATP-driven H -pumping into proteoliposomes
collapsed immediately upon the addition of gra-
micidin D. No proton pumping could be detected
after pretreatment of the proteoliposomes with
KNO3.
3.6. Subunits of ATPase isolated from tonoplast
Molecular weight of polypeptides in the reconstituted ATPase was estimated by SDS-PAGE. As
shown in Fig. 7, the reconstituted proteoliposomes
contained three major bands of polypeptides (68,
60 and 16 kDa), in addition to several minor
bands.
4. Discussion
The method used for the isolation of tonoplast
H-ATPase from pumpkin cotyledons (Suzuki
and Kasamo, 1993) was successfully applied to
T. virginiana. H -ATPase isolated from T.
virginiana leaf cells showed sufficiently high activity both in intact tonoplast and in reconstituted
proteoliposomes.
H -ATPase in T. virginiana tonoplast was
purified to 12-fold, as estimated from the increase
in the specific activity (Table 1). The purity of H ATPase was further checked by SDS-PAGE.
Referring to the data of H -ATPase from other
plant material, principal three subunits (shown in
Fig. 7) were speculated as the components of H ATPase. Other minor components, however, were
suspected to be impurity. Tonoplast ATPase has a
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M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
Fig. 1. Effect of inhibitors on ATPase activity of the isolated tonoplasts. Activities without inhibitor were 13.5 mmol Pi per mg protein
h 1. (A) DES, (B) DCCD, (C) NaN3, (D) Oligomycin, (E) Na3VO4, (F) KNO3.
functional unit of 650 kDa (Mandala and Taiz,
1985) being composed of three to nine different
subunits. The catalytic site may possibly be on a
67 /73 kDa polypeptide (Mandala and Taiz, 1986;
Randall and Sze, 1987). Another subunit of 55/62
kDa may perform a regulatory role, since it is
capable of binding adenine nucleotides with high
affinity (Manolson et al., 1985). The third subunit
is a 13 /16 kDa subunit, which is a DCCD-binding
protein and may mediate the passage of protons.
M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
Fig. 2. ATP-dependent quenching of quinacrine /fluorescence
with isolated tonoplasts. The assay medium for the fluorescence
quenching contained 100 mg protein. ATP /MgSO4 was added 5
min after the addition of either inhibitor; 50 mM Na3VO4, 50
mM KNO3.
Fig. 3. Effect of phospholipid (asolectin) to protein ratio on the
ATPase activity in reconstituted proteoliposomes. Reconstitution was carried out in 10 mM MES/Tris (pH 7.2), 100 mM KCl
and 1 mM EDTA with various phospholipid/protein ratios.
ATPase activity was measured for 30 min at pH 7.5 in 3 mM
ATP /MgSO4, 50 mM KCl and 30 mM MES/Tris.
Besides these three principal components, there are
several polypeptides with molecular weights from
13 to 41 kDa. Among the subunits of tonoplast
ATPase, the 13/16 kDa subunit is an integral
membrane protein and the others are associated
peripherally with the tonoplast. The presence of
multiple subunits renders the ATPases more
227
Fig. 4. Effect of KNO3 on the ATPase activity in reconstituted
proteoliposomes. ATPase activity was determined in an assay
buffer containing 3 mM ATP /MgSO4, 50 mM KCl and 30 mM
MES/Tris (pH 7.5). The reaction was initiated by the addition
of reconstituted proteoliposomes containing 20 mg protein. The
specific activity of the ATPase in the reconstituted proteoliposomes was 170 mmol Pi per mg protein h 1.
Fig. 5. pH dependence on the ATPase activity in reconstituted
proteoliposomes. ATPase activity was determined in an assay
buffer containing 3 mM ATP /MgSO4, 50 mM KCl and 30 mM
MES/Tris (pH 7.5). The pH of the assay buffer was adjusted by
varying the Tris to MES ratio. The reaction was initiated by the
addition of reconstituted proteoliposomes containing 20 mg
protein.
similar to F1F0-type ATPases than to the P-type
ATPases.
According to the former studies, H -translocating ATPase are classified into three types; F-, Pand V-types (Pedersen and Carafoli, 1987). In
recent papers, there are many experiments using
bafilomycin A as a specific inhibitor of V-ATPase.
In this paper, however, we have only investigated
whether H-ATPase exists in the tonoplast of T.
virginiana L. leaf cells. Then its properties were
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M. Saito et al. / Journal of Biotechnology 100 (2003) 221 /229
Fig. 6. ATP-dependent quenching of quinacrine /fluorescence
with reconstituted proteoliposomes. Three millimolar ATP /
MgSO4 was added 5 min after the addition of either inhibitor;
50 mM Na3VO4 or 50 mM KNO3.
related to acidic compartments of animal cells
(Forgac, 1989). It is now well established that the
tonoplast is equipped with ATPase in various
tissues of higher plants, such as red-beet root
(Bennett et al., 1984; Poole et al., 1984; Rea et al.,
1987), corn coleoptile (Mandala and Taiz, 1985),
corn root (Bennett and Spanswick, 1983), Tulipa
petal (Wagner and Lin, 1982), oat root (Randall
and Sze, 1986), mung bean hypocotyl (Yoshida et
al., 1986), mung bean root (Kasamo, 1986) and
rice cells in culture (Kasamo, 1988). The role of the
V-type ATPase is understood as the promotion of
the cell growth by generating the electrochemical
potential gradient across the tonoplast. This
potential gradient can provide the driving force
for solute transport, ion uptake and the regulation
of intracellular pH and several other essential
functions.
This study provides direct evidence that tonoplast ATPase incorporated into liposomes catalyzes the pumping of protons across the membrane
of the proteoliposomes. This has certified the
existence of H-translocating ATPase in the
tonoplast of T. virginiana leaf cells.
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Fig. 7. SDS-PAGE (12.5%) of the solubilized ATPase and
reconstituted ATPase. The gels were stained with silver. (A)
Solubilized ATPase, (B) reconstituted ATPase. Numbers on the
side indicate molecular weights in kDa.
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