Vol. 168, No. 3, 1990 May 16, 1990
OLIGOMERIC
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1157-1162
STRUCTURE OF H+-TRANSLOCATING INORGANIC PYROPHOSPHATASE OF PLANT VACUOLES
Masayoshi MAESHIMA Institute
of Low Temperature Science, Hokkaido University,
Received April
Sapporo 060, Japan
6, 1990
The topography and oligomeric structure of the vacuolar membrane-bound inorganic pyrophosphatase (73,000 daltons) of mung bean were studied. When the vacuolar membraneswere treated with thiocyanate or sodium carbonate which are known to remove the peripheral membraneproteins, the enzyme could not be detected in the solubilized fraction by the specific antibody. The apparent molecular size of the enzyme was estimated to be about 480 kDa by polyacrylamide gel electrophoresis in the presence of Triton X-100. Crosslinking treatment of the pyrophosphatase with dimethyl suberimidate produced a complex corresponding to the dimer. The rate of PP. hydrolysis showed a sigmoidal relationship to substrate concentration wit& a Hill coefficient of 2.5. These results suggest that the vacuolar pyrophosphatase is an integral membraneprotein and functions as an oligomer, probably a dimer. 01990 Academic Press,
Inc.
The vacuolar inorganic
pyrophosphatase (EC, 3.6.1.1,
a proton pumpwith the reaction is
purified a
being coupled with the hydrolysis
responsible for generating an internal
and a proton
gradient
of 73 kDa (2).
cells
(1,7).
understand the relationship
protein,
this
Recently
the
the
concerning the topology
the
role
in
in
the
of this new proton pump is needed to
between the structure
structure
vacuole
is very simple like
and its
function.
work, the vacuolar PPase was shown to be an integral
and the oligomeric
and
enzyme was
Vacuolar PPase plays an important
structure
as
and demonstrated to consist of
constituent
Biochemical information
membrane and the higher-order
In
Its
of PPi,
environment in
across the membrane(1).
plasma membraneH+-ATPase (5,6). plant
acidic
from mung bean (2) and red beet (3,4),
single polypeptide
PPase) functions
of the purified
membrane
enzyme was analyzed
in
various experiments. 0006-291X00$1.50 1157
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Plant material. Seeds of mung bean (Vigna radiata) were imbibed with water and allowed to germinate in the dark at 26°C for 3.5 days. Hypocotyls were used. Enzyme purification. The vacuolar membraneswere prepared by differential and floating centrifugations (2). The membraneswere treated with 0.3% sodium deoxycholate in 50 mMKC1 to remove some peripheral proteins. The vacuolar PPase was solubilized with 0.4% lysophosphatidylcholine and purified by double QAE-Toyopearl column chromatography (2). Measurements. The activities of PPase (2), the vacuolar ATPase (8) and catalase Protein content was (9) were assayed as described previously. determined by the method of Lowry et al. (10). Immunoblotting. Immunoblotting was performed as described previously (2) by a modified version of the method of Towbin et al. (11). The antibody that reacted with the antigen on a nitrocellulose filter was detected using horseradish peroxidase-linked protein A. Gel electrophoresis on a 12% Polyacrylamide gel electrophoresis. polyacrylamide gel or a 5-15% gradient gel containing 0.1% SDS was carried out by the method of Laemmli (12). Polyacrylamide gradient gel electrophoresis was done in the presence of 0.1% Triton X-100 or 0.1% Lubrol PX in a 4-15% linear gradient gel by a modified version of the method of Tomida et al. (13). Instead of nonaethyleneglycol dodecyl ether, Triton X-100 or Lubrol PX was added to the gel and electrode buffers. MgSOCros;-linking. The purified PPase was dialyzed against 30 mMKCl, 2 mM 5, glycerol and 0.1% Triton X-100. The enzyme (20 pg) was mixed with 0.7 4& of 0.2 M potassium phosphate buffer, 1 mM pH 8.2, containing dithiothreitol, 1 mMMgS04, 10%glycerol, 20 mMtriethanolamine and 37 mM dimethyl suberimidate. After incubation at 25°C for 1 hr, 70 ~1 of 1 M lysine was added to stop the reaction, and the same volume of cold 50% trichloroacetic acid was added to precipitate the proteins. The precipitate was washed with 10%trichloroacetic acid and acetone, and dissolved in the dissociation buffer for SDS/polyacrylamide electrophoresis.
RESULTS AND DISCUSSION The vacuolar PPase which consists of a polypeptide to
The present work offered
membraneprotein.
evidence that the PPase is
vacuolar 1).
hydrophobic released observation the
ATPase was recovered in both the
the multi-subunit
enzymes.
precipitate
the
Recent papers have reported the
membraneby treatment with SCN-'
agrees with these reports.
ability
to
that
the
68-kDa subunit, (14,15).
On the other hand, in
vacuolar PPase was detected only in the precipitate,
PPase is associated directly
and supernatant
anion which has the
complex of vacuolar ATPase, including
from
were
the major subunit (68-kDa subunit) of
SCN-1 is known to be a caotropic
dissociate
an integral
When the vacuolar membranesfrom mung bean hypocotyls
treated with 0.5 M KSCNand centrifuged,
(Fig.
expected
span the vacuolar membrane, since it operates as a proton-translocating
machinery.
the
of 73 kDa is
The present immunoblotting,
suggesting that
with the membraneby its hydrophobic part. 1158
was
the Other
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
234567
1
RESEARCH COMMUNICATIONS
100
2
50
E z
0
m a loo .z z z a 50
0
1
gel
0
0
5 Fraction
2
immunoblot
10
15
number
Figure 1. Treatment of the vacuolar membrane with KSCN. The vacuolar membrane fraction (2 mg/ml) was mixed with the same volume of 1 M KSCN dissolved in 20 mM Tris/acetate, pH 7.5, and incubated at 4'C for 30 min. After were centrifugation at 170,OOOg for 40 min, the precipitate and supernatant subjected to SDS/polyacrylamide gel electrophoresis. Lane 1, vacuolar membrane; lanes 2, 4 and 6, precipitate; lanes 3, 5 and 7, supernatant. Lanes 1-3, Coomassie blue-stained gel. Lanes 4 and 5, immunoblot with the antibody to the largest subunit (68-kDa subunit) of vacuolar ATPase. Lanes 6 and 7, immunoblot with the antibody to the vacuolar PPase. The arrowheads (a and b) indicate the vacuolar PPase and the 68-kDa subunit of ATPase, respectively. Figure 2. Glycerol density gradient centrifugation. The solubilized fraction of vacuolar membranes by lysophosphatidylcholine (A) and the purified PPase 20 mM to 15-ml linear glycerol gradients containing (B) were applied Tris/acetate, ";,,I,~~,~,~ ~f%&'(y di'h~~~~~~~~~v~n~c~~~as~d(so~03~ Triton X-100. 28eFL The gradients 11.30) were mixed with the enzyme solut%"is standards. centrifuged at 80,OOOg for 45 hr at 4'C, and then divided into 0.8-ml The activities of PPase (O), vacuolar ATPase (0) and catalase fractions. and the amount of serum albumin (A) are expressed as the percentages (0) relative to those in the peak fractions.
treatments
such
as sonication
mM Na2C03
failed
to release
previously
(2),
described of bile
salt,
has a very
findings
show that
alkaline
the PPase
the PPase
is
or Tween
80.
X-100
and
activity
These
Triton
under
hydrophobic the
condition
from not
and incubation
the membranes solubilized
(not
domain
in
vacuolar
PPase
profile
of the
its
is
As
concentration
phospholipid
amino-terminal
a typical
100
shown).
by a low
The enzyme requires
in
for
part
integral
(2).
membrane
protein. Fig. density PPase
2 shows gradient
sedimented
the
activity
centrifugation slightly
faster
in the than
presence the
1159
bovine
vacuolar
PPase
of 0.03% serum
Triton
albumin
after
glycerol X-100.
(67 kDa).
The This
BIOCHEMICAL
Vol. 168, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
suggests a monomeric form for PPase based on the assumption that
the
enzyme
contains only small amounts of detergents and/or lipids. Polyacrylamide of detergent faster
gave a single,
in Triton
the migration in
gel electrophoresis
PPase in the
sharp band as shown in Fig. 3.
presence
The PPase migrated
X-100 than in Lubrol PX, although there was no difference
rate of the marker proteins
in
Difference
between the two systems.
the size of PPase between the two systems may be due to the difference
the
bound detergent.
amount of
calculated
massesof micelles
and 64 kDa, respectively much larger
(16).
gradient
kDa was observed
in addition
the
property
PPi
coefficient
vacuolar
PPase functions Cross-linking
dimethyl
suberimidate
to the 73-kDa band (Fig.
hydrolysis
is
a
SDS/polyacrylamide
This
4).
showed a sigmoidal and the Hill
relationship
coefficient
158
finding
was 2.0 oligomeric
study using the purified
(not
shown).
(dimeric)
translocation The allosteric
enzyme, I propose that
the
vacuolar
73-kDa subunit.
enzyme suggests a dimeric form for
as an enzyme-detergent complex, a large
PPase.
3) and in the apparent partial
Since
the functional
specific
size
volume of the enzyme (Fig.
form of the plasma membraneATPase which consists 1160
As
amount of
detergent bound to the enzyme causes the marked increase in the apparent (Fig.
the
structure,
in the membraneas an oligomer comprised of of the purified
to
was 2.4 (Fig.
for the rate of PPi-dependent proton
membrane vesicles
vacuolar PPase exists
which
for PPase.
of PPase also supports its
From the
the
of
with
(PPi plus Mg2+) concentration
The Hill
a
PPase, the
a broad band with a molecular mass of about
suggests a dimeric structure
5).
were
the PPase had been composedof
When the products were subjected to
gel electrophoresis,
substrate
experiment
(73 kDa).
reagent.
The rate
The
90 kDa
X-100 and Lubrol PX are about
The values obtained in this
PPase was cross-linked
bifunctional
PPase was
to determine the subunit number of the functional
order
purified
of Triton
in
X-100 or 600 kDa in Lubrol PX.
than would have been expected if
single polypeptide In
The apparent molecular mass of
to be about 480 kDa in Triton
molecular
of
of the purified
2). of
a
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
10 6-
4-
2-
-1-
V
Vmax- V
2320.4 -
0.2 -
-
0
67-
0.11 0.1
5
Gl
Lubrol
PX
Triton
X-100
S
0.2tmM)
0.4
Figure 3. Polyacrylamide gradient gel electrophoresis in the presence of detergent. Electrophoresis was done in 4-15% gradient gel containing 0.1% Lubrol PX (lanes 1 and 2) or 0.1% Triton X-100 (lanes 3 and 4). The gel was stained with Coomassie blue. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa) were used as standard proteins (lanes 1 and 3). Lanes 2 and 4, purified PPase. Figure 4. Gel electrophoresis of the vacuolar PPase after cross-linking. The purified enzyme was cross-linked for 1 hr at 25'C with dimethyl suberimidate as described in "Materials and Methods." Samples were separated in a S-15% gradient Laemmli gel. Lane 1, purified PPase; lane 2, cross-linked product. Molecular masses (kDa) of standards are given on the left. Hill analysis of the dependence of PPase activity on substrate Figure 5. concentration. The reaction mixture contained 30 mM Tris/Mes, pH 7.2, 50 mM KCl, 0.02% Triton X-100, 1 mM sodium molybdate, and equal concentrations of PPi and MgS04 as indicated. Vmax was 23 nmolfmin.
single
polypeptide
topography primary
of about
of vacuolar structure
proton-translocating
100 kDa was thought
PPase may be similar of PPase needs enzymes
which
to be that
to the
to be determined have
been studied
plasma
of a dimer membrane
to compare at the
it
(5),
the
ATPase.
The
with
molecular
other level.
ACKNOWLEDGMENTS. This study was supported by Grants-in-Aid for Scientific Research (No. 01560080) and Scientific Research on Priority of "Bioenergetics" from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Boller, T., and Wiemken, A. (1986) Annu. S. (1989) J. Biol. 2. Maeshima, M., Yoshida, 1161
Rev. Plant Physiol. 37, Chem. 264, 20068-20073.
137-164.
0.6
Vol.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Sarafian, V., and Poole, R.J. (1989) Plant Physiol.91, 34-38. Britten, C.J., Turner, J.C., and Rea, P.A. (1989) FEBSLett. 256, 200-206. Bowman,B.J., and Bowman,E.J. (1986) J. Membrane Biol. 94, 83-97. Serrano, R. (1988) Biochim. Biophys. Acta 947, l-28. Maeshima, M. (1990) Plant Cell Physiol. 31, (in press). Matsuura-Endo, C., Maeshima, M., and Yoshida, S. (1990) Eur. J. Biochem. 187, 745-751. Luck, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed. 1 pp.885-894, Acadexc Press, New York. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-273. Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci U.S.A. 76, 4350-4354. Laemmli, U.K. (1970) Nature 227, 680-683. Tomida, M., Kondo, Y., Moriyama, R., Machida, H., and Makino, S. (1988) Biochim. Biophys. Acta 943, 493-500. Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582. Parry, R.V., Turner, J.C., and Rea, P.A. (1989) J. Biol. Chem. 264, 2002520032. Hjelmeland, L.M. (1986) Methods Enzymol. 124, 135-164.
1162
OLIGOMERIC
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1157-1162
STRUCTURE OF H+-TRANSLOCATING INORGANIC PYROPHOSPHATASE OF PLANT VACUOLES
Masayoshi MAESHIMA Institute
of Low Temperature Science, Hokkaido University,
Received April
Sapporo 060, Japan
6, 1990
The topography and oligomeric structure of the vacuolar membrane-bound inorganic pyrophosphatase (73,000 daltons) of mung bean were studied. When the vacuolar membraneswere treated with thiocyanate or sodium carbonate which are known to remove the peripheral membraneproteins, the enzyme could not be detected in the solubilized fraction by the specific antibody. The apparent molecular size of the enzyme was estimated to be about 480 kDa by polyacrylamide gel electrophoresis in the presence of Triton X-100. Crosslinking treatment of the pyrophosphatase with dimethyl suberimidate produced a complex corresponding to the dimer. The rate of PP. hydrolysis showed a sigmoidal relationship to substrate concentration wit& a Hill coefficient of 2.5. These results suggest that the vacuolar pyrophosphatase is an integral membraneprotein and functions as an oligomer, probably a dimer. 01990 Academic Press,
Inc.
The vacuolar inorganic
pyrophosphatase (EC, 3.6.1.1,
a proton pumpwith the reaction is
purified a
being coupled with the hydrolysis
responsible for generating an internal
and a proton
gradient
of 73 kDa (2).
cells
(1,7).
understand the relationship
protein,
this
Recently
the
the
concerning the topology
the
role
in
in
the
of this new proton pump is needed to
between the structure
structure
vacuole
is very simple like
and its
function.
work, the vacuolar PPase was shown to be an integral
and the oligomeric
and
enzyme was
Vacuolar PPase plays an important
structure
as
and demonstrated to consist of
constituent
Biochemical information
membrane and the higher-order
In
Its
of PPi,
environment in
across the membrane(1).
plasma membraneH+-ATPase (5,6). plant
acidic
from mung bean (2) and red beet (3,4),
single polypeptide
PPase) functions
of the purified
membrane
enzyme was analyzed
in
various experiments. 0006-291X00$1.50 1157
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Plant material. Seeds of mung bean (Vigna radiata) were imbibed with water and allowed to germinate in the dark at 26°C for 3.5 days. Hypocotyls were used. Enzyme purification. The vacuolar membraneswere prepared by differential and floating centrifugations (2). The membraneswere treated with 0.3% sodium deoxycholate in 50 mMKC1 to remove some peripheral proteins. The vacuolar PPase was solubilized with 0.4% lysophosphatidylcholine and purified by double QAE-Toyopearl column chromatography (2). Measurements. The activities of PPase (2), the vacuolar ATPase (8) and catalase Protein content was (9) were assayed as described previously. determined by the method of Lowry et al. (10). Immunoblotting. Immunoblotting was performed as described previously (2) by a modified version of the method of Towbin et al. (11). The antibody that reacted with the antigen on a nitrocellulose filter was detected using horseradish peroxidase-linked protein A. Gel electrophoresis on a 12% Polyacrylamide gel electrophoresis. polyacrylamide gel or a 5-15% gradient gel containing 0.1% SDS was carried out by the method of Laemmli (12). Polyacrylamide gradient gel electrophoresis was done in the presence of 0.1% Triton X-100 or 0.1% Lubrol PX in a 4-15% linear gradient gel by a modified version of the method of Tomida et al. (13). Instead of nonaethyleneglycol dodecyl ether, Triton X-100 or Lubrol PX was added to the gel and electrode buffers. MgSOCros;-linking. The purified PPase was dialyzed against 30 mMKCl, 2 mM 5, glycerol and 0.1% Triton X-100. The enzyme (20 pg) was mixed with 0.7 4& of 0.2 M potassium phosphate buffer, 1 mM pH 8.2, containing dithiothreitol, 1 mMMgS04, 10%glycerol, 20 mMtriethanolamine and 37 mM dimethyl suberimidate. After incubation at 25°C for 1 hr, 70 ~1 of 1 M lysine was added to stop the reaction, and the same volume of cold 50% trichloroacetic acid was added to precipitate the proteins. The precipitate was washed with 10%trichloroacetic acid and acetone, and dissolved in the dissociation buffer for SDS/polyacrylamide electrophoresis.
RESULTS AND DISCUSSION The vacuolar PPase which consists of a polypeptide to
The present work offered
membraneprotein.
evidence that the PPase is
vacuolar 1).
hydrophobic released observation the
ATPase was recovered in both the
the multi-subunit
enzymes.
precipitate
the
Recent papers have reported the
membraneby treatment with SCN-'
agrees with these reports.
ability
to
that
the
68-kDa subunit, (14,15).
On the other hand, in
vacuolar PPase was detected only in the precipitate,
PPase is associated directly
and supernatant
anion which has the
complex of vacuolar ATPase, including
from
were
the major subunit (68-kDa subunit) of
SCN-1 is known to be a caotropic
dissociate
an integral
When the vacuolar membranesfrom mung bean hypocotyls
treated with 0.5 M KSCNand centrifuged,
(Fig.
expected
span the vacuolar membrane, since it operates as a proton-translocating
machinery.
the
of 73 kDa is
The present immunoblotting,
suggesting that
with the membraneby its hydrophobic part. 1158
was
the Other
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
234567
1
RESEARCH COMMUNICATIONS
100
2
50
E z
0
m a loo .z z z a 50
0
1
gel
0
0
5 Fraction
2
immunoblot
10
15
number
Figure 1. Treatment of the vacuolar membrane with KSCN. The vacuolar membrane fraction (2 mg/ml) was mixed with the same volume of 1 M KSCN dissolved in 20 mM Tris/acetate, pH 7.5, and incubated at 4'C for 30 min. After were centrifugation at 170,OOOg for 40 min, the precipitate and supernatant subjected to SDS/polyacrylamide gel electrophoresis. Lane 1, vacuolar membrane; lanes 2, 4 and 6, precipitate; lanes 3, 5 and 7, supernatant. Lanes 1-3, Coomassie blue-stained gel. Lanes 4 and 5, immunoblot with the antibody to the largest subunit (68-kDa subunit) of vacuolar ATPase. Lanes 6 and 7, immunoblot with the antibody to the vacuolar PPase. The arrowheads (a and b) indicate the vacuolar PPase and the 68-kDa subunit of ATPase, respectively. Figure 2. Glycerol density gradient centrifugation. The solubilized fraction of vacuolar membranes by lysophosphatidylcholine (A) and the purified PPase 20 mM to 15-ml linear glycerol gradients containing (B) were applied Tris/acetate, ";,,I,~~,~,~ ~f%&'(y di'h~~~~~~~~~v~n~c~~~as~d(so~03~ Triton X-100. 28eFL The gradients 11.30) were mixed with the enzyme solut%"is standards. centrifuged at 80,OOOg for 45 hr at 4'C, and then divided into 0.8-ml The activities of PPase (O), vacuolar ATPase (0) and catalase fractions. and the amount of serum albumin (A) are expressed as the percentages (0) relative to those in the peak fractions.
treatments
such
as sonication
mM Na2C03
failed
to release
previously
(2),
described of bile
salt,
has a very
findings
show that
alkaline
the PPase
the PPase
is
or Tween
80.
X-100
and
activity
These
Triton
under
hydrophobic the
condition
from not
and incubation
the membranes solubilized
(not
domain
in
vacuolar
PPase
profile
of the
its
is
As
concentration
phospholipid
amino-terminal
a typical
100
shown).
by a low
The enzyme requires
in
for
part
integral
(2).
membrane
protein. Fig. density PPase
2 shows gradient
sedimented
the
activity
centrifugation slightly
faster
in the than
presence the
1159
bovine
vacuolar
PPase
of 0.03% serum
Triton
albumin
after
glycerol X-100.
(67 kDa).
The This
BIOCHEMICAL
Vol. 168, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
suggests a monomeric form for PPase based on the assumption that
the
enzyme
contains only small amounts of detergents and/or lipids. Polyacrylamide of detergent faster
gave a single,
in Triton
the migration in
gel electrophoresis
PPase in the
sharp band as shown in Fig. 3.
presence
The PPase migrated
X-100 than in Lubrol PX, although there was no difference
rate of the marker proteins
in
Difference
between the two systems.
the size of PPase between the two systems may be due to the difference
the
bound detergent.
amount of
calculated
massesof micelles
and 64 kDa, respectively much larger
(16).
gradient
kDa was observed
in addition
the
property
PPi
coefficient
vacuolar
PPase functions Cross-linking
dimethyl
suberimidate
to the 73-kDa band (Fig.
hydrolysis
is
a
SDS/polyacrylamide
This
4).
showed a sigmoidal and the Hill
relationship
coefficient
158
finding
was 2.0 oligomeric
study using the purified
(not
shown).
(dimeric)
translocation The allosteric
enzyme, I propose that
the
vacuolar
73-kDa subunit.
enzyme suggests a dimeric form for
as an enzyme-detergent complex, a large
PPase.
3) and in the apparent partial
Since
the functional
specific
size
volume of the enzyme (Fig.
form of the plasma membraneATPase which consists 1160
As
amount of
detergent bound to the enzyme causes the marked increase in the apparent (Fig.
the
structure,
in the membraneas an oligomer comprised of of the purified
to
was 2.4 (Fig.
for the rate of PPi-dependent proton
membrane vesicles
vacuolar PPase exists
which
for PPase.
of PPase also supports its
From the
the
of
with
(PPi plus Mg2+) concentration
The Hill
a
PPase, the
a broad band with a molecular mass of about
suggests a dimeric structure
5).
were
the PPase had been composedof
When the products were subjected to
gel electrophoresis,
substrate
experiment
(73 kDa).
reagent.
The rate
The
90 kDa
X-100 and Lubrol PX are about
The values obtained in this
PPase was cross-linked
bifunctional
PPase was
to determine the subunit number of the functional
order
purified
of Triton
in
X-100 or 600 kDa in Lubrol PX.
than would have been expected if
single polypeptide In
The apparent molecular mass of
to be about 480 kDa in Triton
molecular
of
of the purified
2). of
a
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
10 6-
4-
2-
-1-
V
Vmax- V
2320.4 -
0.2 -
-
0
67-
0.11 0.1
5
Gl
Lubrol
PX
Triton
X-100
S
0.2tmM)
0.4
Figure 3. Polyacrylamide gradient gel electrophoresis in the presence of detergent. Electrophoresis was done in 4-15% gradient gel containing 0.1% Lubrol PX (lanes 1 and 2) or 0.1% Triton X-100 (lanes 3 and 4). The gel was stained with Coomassie blue. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa) were used as standard proteins (lanes 1 and 3). Lanes 2 and 4, purified PPase. Figure 4. Gel electrophoresis of the vacuolar PPase after cross-linking. The purified enzyme was cross-linked for 1 hr at 25'C with dimethyl suberimidate as described in "Materials and Methods." Samples were separated in a S-15% gradient Laemmli gel. Lane 1, purified PPase; lane 2, cross-linked product. Molecular masses (kDa) of standards are given on the left. Hill analysis of the dependence of PPase activity on substrate Figure 5. concentration. The reaction mixture contained 30 mM Tris/Mes, pH 7.2, 50 mM KCl, 0.02% Triton X-100, 1 mM sodium molybdate, and equal concentrations of PPi and MgS04 as indicated. Vmax was 23 nmolfmin.
single
polypeptide
topography primary
of about
of vacuolar structure
proton-translocating
100 kDa was thought
PPase may be similar of PPase needs enzymes
which
to be that
to the
to be determined have
been studied
plasma
of a dimer membrane
to compare at the
it
(5),
the
ATPase.
The
with
molecular
other level.
ACKNOWLEDGMENTS. This study was supported by Grants-in-Aid for Scientific Research (No. 01560080) and Scientific Research on Priority of "Bioenergetics" from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Boller, T., and Wiemken, A. (1986) Annu. S. (1989) J. Biol. 2. Maeshima, M., Yoshida, 1161
Rev. Plant Physiol. 37, Chem. 264, 20068-20073.
137-164.
0.6
Vol.
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