ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 346, No. 1, October 1, pp. 105–112, 1997
Article No. BB970279
Purification and Characterization of Thylakoid
Membrane-Bound Inorganic Pyrophosphatase
from Spinacia oleracia L.
Shih Sheng Jiang, Lin Lin Fan, Su Jing Yang, Soong Yu Kuo, and Rong Long Pan1
Institute of Radiation Biology, College of Nuclear Science, National Tsing Hua University,
Hsin Chu, 30043 Taiwan, Republic of China
Received June 2, 1997
An inorganic pyrophosphatase (PPase) was purified
from thylakoid membrane of spinach leaves to electrophoretic purity by methods including detergent solubilization, ammonium sulfate fractionation, and successive chromatographic techniques. Current protocol
yielded about 10% recovery of total activity with a 30fold purification. The specific activity of the purified
enzyme was approximately 400 mmol PPi consumed/mg
proteinrh. This enzyme is a monomer with a molecular
mass of 55 kDa. Several properties, including subunit
composition, substrate specificity, ion requirements,
inhibitor sensitivities, and amino acid composition,
have been studied. Mg2/ is an essential cofactor for
the thylakoid PPase. The preferred substrate for the
hydrolytic reaction of PPase appears to be dimagnesium pyrophosphate. K/ could not stimulate the enzymatic activity of thylakoid PPase, while F0 was a potent inhibitor. Group-specific modification of the thylakoid PPase demonstrates possible involvement of
carboxylate residues in the enzymatic activity. Furthermore, antibodies raised against thylakoid PPase
in a rabbit could inactivate the PPi hydrolysis of thylakoid and the purified enzyme, but not that of vacuolar
H/-PPase, indicating both PPi hydrolases are structurally distinct. q 1997 Academic Press
Inorganic pyrophosphatase (PPase, EC 3.6.1.1)2
plays a vital role in energy metabolism. The hydrolysis
1
To whom correspondence should be addressed. Fax: (886)(3)5719744. E-mail: [email protected].
2
Abbreviations used: OG, n-octyl b-D-glucopyranoside; PMSF,
phenylmethylsulfonyl fluoride; PPase, pyrophosphatase; FPLC, fast
protein liquid chromatography; BSA, bovine serum albumin; EDC,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; TNM, tetranitromethane; FITC, fluorescein 5*-isothiocyanate.
of PPi by PPases provides driving forces for a wide
range of biosynthetic polymerizations. Various PPases
were found as soluble enzymes in many different subcellular compartments (1). In addition, several PPases
were also observed on membranes of photosynthetic
bacteria (2) and animal mitochondria (3). In higher
plants, vacuoles (4), mitochondria (5), and chloroplasts
(6) contain many distinct PPi hydrolases. The membrane-bound PPase of tonoplast is a novel proton translocating enzyme belonging to its own category (7). The
vacuolar H/-translocating PPase consists of a single
kind of polypeptide with a molecular mass of 73 kDa
(8, 9). The structure and function of vacuolar H/-translocating PPase was recently isolated from peas using
preparative electrophoresis (10). The membrane-bound
PPase of mitochondria is also a monomeric enzyme
with a much lower molecular mass (33 kDa). Some
properties of mitochondrial membrane-bound PPase
were investigated (5).
The presence of membrane-bound inorganic pyrophosphatase has been demonstrated in chloroplast of
higher plants for years (6, 11–13). However, no purification and characterization of thylakoid PPase has
been reported. The knowledge about the identity, subunit composition, physiological function, and structure
of the thylakoid-associated PPase is still lacking. In
this work, we reported for the first time the isolation
and purification of a membrane-bound PPase from thylakoid membrane by methods including detergent solubilization, ammonium sulfate fractionation, and successive chromatographic techniques. Several properties such as subunit composition, substrate specificity,
inhibitor sensitivities, ion requirements, and amino
acid composition were scrutinized. Furthermore, we
raised in a rabbit the antibody against the purified
thylakoid PPase. The PPi hydrolysis of purified and
membrane-bound thylakoid PPase could be inhibited
by the serum containing the antibody.
105
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Copyright q 1997 by Academic Press
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JIANG ET AL.
MATERIALS AND METHODS
Preparation of thylakoid membranes. Spinach leaves (250 g) were
deribbed and homogenized with a Waring blender for 15–20 s in 300
ml of Buffer I containing 0.3 M sucrose, 30 mM Tris–HCl (pH 7.8),
10 mM NaCl, 3 mM MgCl2 , 0.1 mM PMSF, and 0.5 mM EDTA. The
homogenate was filtered through four layers of cheesecloth. The filtrate was centrifuged at 11,000g for 15 min and the pellet resuspended in Buffer I. The resuspension was subjected to differential
centrifugation at 500g for 3 min and at 2300g for 5 min. The pellet
was resuspended in Buffer II containing 20 mM Tris–HCl (pH 7.6),
0.2 M sucrose, and 10 mM NaCl and washed exhaustively (at least
three times) before final precipitation at 13,300g for 1 min. The thylakoid membranes were resuspended in a solution containing an equal
volume of Buffer II and storage buffer [25 mM Tris–HCl (pH 7.5),
10% (v/v) glycerol, and 10 mM NaCl]. All the following isolation procedures except FPLC and HPLC were carried out at 47C.
Purification of pyrophosphatase. Two hundred milligrams of thylakoid membrane was incubated in 300 ml solubilization buffer [50
mM Tris–HCl (pH 8.0), 0.5 mM EDTA, 0.2 mM dithiothreitol, 25%
(v/v) glycerol, and 35 mM octyl glucopyranoside (OG)] to solubilize
membrane-bound PPase. After slow addition of detergent and gentle
stirring for 1 h, the suspension was centrifuged at 130,000g for 2 h.
The pellet was discarded and ammonium sulfate solution was added
to supernatant to 50% saturation under stirring for 30 min. After
centrifugation at 12,000g for 10 min, the pellet containing no PPase
activity was removed. Ammonium sulfate was further added to the
supernatant to 75% saturation. The PPase precipitate was collected
by additional centrifugation at 12,000g for 10 min and resuspended
in Buffer III [50 mM Tris–HCl (pH 7.5), 0.2 mM EDTA, 0.4 mM
dithiothreitol, and 20% (v/v) glycerol]. The suspension was then centrifuged at 12,000g for 10 min to remove insoluble materials. The
final supernatant was then subjected to further chromatography.
The PPase containing suspension above (20 mg total protein) was
applied onto a hydroxyapatite column (1.6 1 20 cm) preequilibrated
with Buffer III and then eluted with the same buffer at a flow rate
of 0.8 ml/min. While pigments were retained on the top of the gel,
the flowthrough containing PPase activity was collected and then
subjected to a buffer change and concentration by ultrafiltration in
an Amicon cell with a YM-10 membrane. After the buffer was exchanged to Buffer IV [20 mM Tris–HCl (pH 7.5), 0.4 mM dithiothreitol, 10% (v/v) glycerol, 50 mM NaCl, and 0.03% (w/v) NaN3], the
concentrate (8 ml) was loaded on a Sephadex G-200 column (2.6 1
65 cm) preequilibrated with Buffer IV. The PPase was eluted by the
equilibrium buffer at a flow rate of 0.25 ml/min. Fractions with the
highest PPase activity were pooled for further chromatography.
The fractions containing PPase activity from Sephadex G-200 were
diluted (1:1) with Buffer V containing 20 mM Tris–HCl (pH 7.5), 0.4
mM dithiothreitol, and 10% (v/v) glycerol. The diluted PPase solution
was applied onto a Mono Q HR 5/5 (Pharmacia) anion-exchange
FPLC column. The PPase was eluted by a linear gradient (25 ml)
from 25 to 400 mM KCl in Buffer V. The fractions containing PPase
activity were pooled and stored at 0707C for further studies. For the
preparation of antibody, a second chromatography on Mono Q was
performed.
Enzyme assay and protein determination. PPase activity was assayed by measuring the enzymatic release of phosphate from inorganic pyrophosphate as determined according to Fiske and Subbarow
(14) with minor modifications. Two to 5 mg of enzyme was assayed
in 1 ml medium containing 1 mM sodium pyrophosphate, 2 mM
MgCl2 , 25 mM Tris–HCl (pH 8.5), 0.1 mM ammonium molybdate
(acid phosphatase inhibitor), 0.2 mM sodium vanadate (P-type
ATPase inhibitor), 0.5 mM sodium azide (F-type ATPase inhibitor),
50 mM potassium nitrate (V-type ATPase inhibitor), and 15 mg/ml
phosphatidylcholine. For the enzyme assay of purified PPase, inhibitors mentioned above were excluded. After incubation at 337C for
10 to 20 min, within which the activity is linear, the reaction was
terminated by adding a 2-ml solution containing 1.7% (w/v) ammo-
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nium molybdate, 2% (w/v) SDS, and 0.02% (w/v) 1-amino-2-naphthol4-sulfonic acid at room temperature. The released Pi was measured
spectrophotometrically at 700 nm. Since 2 mol of Pi was liberated
for each mole of PPi hydrolyzed, the specific activity was expressed
as mmol PPi consumed/mg proteinrh. Each data value was the average of at least three assays with standard deviation less than 10%.
Protein concentration was determined using a protein assay kit
(Bio-Rad) according to Bradford (15) using BSA as the standard. To
measure the native molecular mass, the purified PPase was applied
onto a Protein Pak-125 (Waters) HPLC gel filtration column preequilibrated with a buffer containing 10 mM Tris–HCl (pH 7.5), 0.1 M
K2SO4 , and 0.05% (w/v) NaN3 . Molecular mass standards include
BSA (66 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7
kDa), and vitamin B12 (1.4 kDa). The flow rate of HPLC was 0.75
ml/min.
SDS–PAGE. Gel electrophoresis was carried out either on a
PhastSystem (Pharmacia) using PhastGel gradient 8–25 (Pharmacia) for purity test or on a Bio-Rad Mini Protein II dual slab
cell using 12% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS
according to Laemmli (16) for the purpose of Western blot. Resolved
polypeptides were visualized either by silver or Coomassie brilliant
blue staining.
Preparation of antibody. Antibodies against thylakoid PPase
were raised in a rabbit by injection of enzyme purified by two consecutive rounds of Mono Q chromatography as mentioned above. For
primary immunization, the purified PPase (100 mg) on SDS–PAGE
was sliced and homogenized with a tissue homogenizer in 2 ml complete Freund’s adjuvant and injected subcutaneously to a rabbit at
multiple sites. For each subsequent boost, the same amount of enzyme was homogenized completely in 1.5 ml incomplete Freund’s
adjuvant oil and then subjected to injection. Serum collected (2 weeks
after boost) without further purification was used for immunoblot
assay.
Immunoblot assay. Following SDS–PAGE, the purified PPase
was transferred onto a nitrocellulose membrane by electroblotting
at 100 mA for 1.5 h in TBE buffer [50 mM Tris–HCl (pH 8.2), 50
mM boric acid, and 1 mM EDTA]. The membranes were soaked for
1 h at room temperature in TTBS buffer [20 mM Tris–HCl (pH 7.4),
0.5 mM NaCl, 0.05% (v/v) Tween 20, and 5% (w/v) skim milk]. The
blotted proteins were allowed to react with sera diluted (1:9000) in
TTBS buffer. The bound antibodies were visualized by a goat antirabbit horseradish peroxidase conjugated IgG.
Amino acid composition. PPase was precipitated with 80% (w/v)
acetone and washed several times for amino acid composition assay,
which was performed on a Millipore Pico Tag system at Institute of
Life Science, National Tsing Hua University. The content of tryptophan was determined by the method of Spandee and Witkop (17).
RESULTS AND DISCUSSION
Purification of Thylakoid PPase
First of all, we determined the purity of the thylakoid
membrane. After exhaustive wash of thylakoid membranes by Buffer II, the PPi hydrolysis activity in supernatant was negligible. Immunoassay (see below) also
showed a negative reaction of above supernatant fractions toward anti-thylakoid PPase antibody (data not
shown), indicating the exclusion of any soluble PPihydrolases and general phosphatases from our starting
membrane preparations. The contamination by other
organelles, such as ER, Golgi bodies, vacuolar vesicles,
and mitochondria, as determined by respective marker
enzymes (18–20), was very minor. The purified PPase
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THYLAKOID PYROPHOSPHATASE
FIG. 1. Solubilization of pyrophosphatase from thylakoid membrane by OG. Thylakoid membranes were treated with various concentrations of OG for 1 h. The pyrophosphatase activity was then
assayed in the supernatant (s) and the pellet (l) after centrifugation
at 130,000g for 2 h. The protein concentration and the enzymatic
reaction were measured as described under Materials and Methods.
from thylakoid membranes isolated by Percoll gradient
(21) and the large-scale protocol described under Materials and Methods was very similar as tested by immunoassay. Furthermore, the antibody against thylakoid
PPase failed to recognize any protein from isolated organelles mentioned above, confirming that our PPase
is solely from thylakoid membrane (data not shown).
The thylakoid membranes routinely prepared by the
above-mentioned methods were thus used for this
work. Detailed purification procedures are as described
under Materials and Methods. Among several detergents scrutinized, only OG solubilized PPase activity
effectively from thylakoid membranes. Ionic detergents
(deoxycholate and cholate), organic solvents (chloroform and acetone), and Triton X-100 were less effective.
At an OG concentration of 35 mM, over 95% PPase
activity along with some other thylakoid proteins was
solubilized from thylakoid membranes (Fig. 1). After
ammonium sulfate fractionation of OG-solubilized proteins, the PPase suspension was still contaminated by
chloroplast pigments. Upon hydroxylapatite chromatography, pigments were retained on the top of the gel.
PPase activity was found in the clear flowthrough. No
detectable PPase activity was observed in the fractions
later eluted from the hydroxylapatite column.
The proteins from the flowthrough of hydroxyapatite
chromatography were concentrated and applied onto a
preparative Sephadex G-200 column. The chromatogram demonstrates that a minor protein peak around
55 kDa contains the PPase activity. After gel filtration,
aliquots with the PPase activity were pooled and further purified on a Mono Q FPLC column. The PPase
activity was eluted at a KCl concentration of 250 mM
(Fig. 2). To ensure the purity of PPase, a second Mono-
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107
Q chromatography was performed. A predominantly
single peak of second Mono-Q chromatography confirmed the high purity of the enzyme obtained according to current methods.
The purification of thylakoid PPase is summarized
in Table I. This protocol yielded about 10% recovery of
total activity with a 30-fold purification. The specific
activity of the purified enzyme was approximately 400
mmol PPi consumed/mg proteinrh. The accurate molecular mass of thylakoid PPase was determined on both
HPLC and Suprose 12 FPLC columns as a 55-kDa protein. Upon SDS–PAGE (Fig. 3), the purified protein
migrated as a sharp single band with a molecular mass
of 55 kDa. Further radiation inactivation analysis indicated that the functional masses of thylakoid PPases
on membrane and in the purified form fall in the range
of 55 to 65 kDa (data not shown). We conclude that the
purified PPase is presumably in a monomeric form of
55 kDa. This molecular mass is similar to that of H/PPi synthase from chromatophore of Rhodospirillum
rubrum (56 kDa (22)) but different from those of proton
translocating vacuolar PPase (73 kDa (8)) and some
soluble PPi-hydrolases (20 to 40 kDa) found in the chloroplast (13, 23–25), chromoplast (26), Escherichia coli
(27), and yeast (28).
Characterization of Purified Thylakoid PPase
Purified PPase showed a pH optimum at 8.5–9.0,
in a good agreement with preliminary observation on
PPase activities of thylakoid preparations elsewhere
(6, 12). The thylakoid PPase is likely an alkaline inorganic pyrophosphatase. All measurements were thus
routinely done at pH 8.5. Table II shows that thylakoid
PPase preferentially used PPi as its substrate. P-nitrophenol phosphate, a common substrate for general
FIG. 2. Elution of purified thylakoid pyrophosphatase from MonoQ chromatography. Twenty microliters of each fraction was withdrawn for enzyme assay (l). The chromatographic and reaction conditions were as described under Materials and Methods. Absorbance
of protein was recorded directly from UV/vis is spectrophotometer
monitor (—).
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JIANG ET AL.
TABLE I
Purification of PPase from Spinach Thylakoid Membrane
PPase activity
Total protein
(mg (%))
Specific activity
(mmol PPi /mg.h)
Total activity
(mmol PPi /h)
Recovery
(%)
Fold
purification
Crude
AS
HA
917 (100)
146 (16)
90 (10)
14
24
30
12618
3480
2676
100
28
21
1
1.5
2.1
G-200
MonoQ
21 (2)
3 (0.3)
107
405
2286
1215
18
10
7.8
29.3
Fraction
Note. Thylakoids were solubilized and pyrophophatase purified as described in the text. Reaction conditions were as described under
Materials and Methods. Recovery is the ratio of total activity in each step to that of the thylakoid membrane (1100). AS, ammonium sulfate
precipitate. HA, hydroxyapatite chromatography.
phosphatases, was not hydrolyzed effectively by thylakoid PPase. The possibility that our purified enzyme
was contaminated by acid phosphatase was thus excluded. Nucleotides were relatively poor substrates for
thylakoid PPase. Thylakoid PPase could not hydrolyze
glucose 6-phosphate nor phospho-containing amino
acids such as phosphoserine and phosphothreonine.
PPase activity increases hyperbolically with increasing
PPi concentrations up to 1 mM. However, a higher concentration of PPi was inhibitory to PPase activity (data
not shown). It was therefore not possible to calculate
the Km value of the thylakoid PPase. The inhibitory
effect of the substrate might be due to the fact that
FIG. 3. SDS–PAGE of purified thylakoid pyrophosphatase. Lanes
1 and 8, the standard markers with molecular masses indicated on
the left; lanes 2–7, the preparations from thylakoid membrane, ammonium sulfate precipitate, hydroxyapatite column, G-200 gel filtration, and first and second Mono-Q chromatographies, respectively.
The amounts of proteins loaded on each lane of Phastsystem electrophoresis apparatus were 80 (lane 2–5) and 20 (lanes 6 and 7) ng,
respectively. The preparation of the samples and the electrophoresis
conditions were as described under Materials and Methods.
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excess PPi may act as a chelator of indispensable cofactor Mg2/ (see below). Alternatively, we can not exclude
the possibility that the enzyme contains an additional
PPi binding site for the regulation of its activity. A
similar phenomenon had also been found for vacuolar
H/-PPase isolated from tonoplast membrane of mung
bean seedlings (9).
Effects of several detergents (deoxycholate and Triton X-100) and phospholipids (phosphatidylcholine and
lysophosphatidylcholine) on the purified thylakoid
PPase were scrutinized (data not shown). The reaction
of thylakoid PPase was slightly stimulated (approx
20%) by phospholipids at concentrations of 100 mg/ml
and detergents at 0.05% (w/v), respectively. The poor
requirement of phospholipids and detergent micelles
for the enzymatic activity suggests that thylakoid
PPase might be a peripheral enzyme rather than a
transmembrane protein complex like the vacuolar H/PPase (cf. 9). Thylakoid PPase absolutely required
Mg2/ for its enzymatic activities (Table III). Monovalent cations were not cofactors for enzymatic activity;
neither were other divalent cations such as Ca2/, Cd2/,
Co2/, and Cu2/. The concentration effect of Mg2/ was
further investigated (Fig. 4). When the PPi concentration was fixed at 2 mM, PPase activity was negligible
until Mg2/ concentration reached 0.5 mM. PPase activity then increases sigmoidally with respect to a total
Mg2/ concentration, reflecting a possible cooperative
binding of magnesium to enzyme. Unlike vacuolar
PPase (9), a further increase in Mg2/ concentration did
not inhibit activity of the thylakoid PPase. The optimal
PPase activity was obtained at a Mg2/:PPi ratio of 2:1.
The true substrate of thylakoid PPase might be dimagnesium pyrophosphate as suggested for vacuolar H/PPase (cf. 29).
The enzymatic activity of thylakoid PPase could be
completely diminished by F0, a common inhibitor of
most PPases from a variety of sources. K/ could not
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THYLAKOID PYROPHOSPHATASE
TABLE II
Substrate Specificity of Thylakoid PPase
Specific activity
(mmol PPi /mg proteinrh (%))
Substrate
PPi
ATP
GTP
CTP
UTP
ITP
356.0
3.9
51.3
43.8
12.1
29.2
{
{
{
{
{
{
18.0
0.2
4.1
2.2
1.4
5.8
Specific activity
(mmol PPi /mg proteinrh (%))
Substrate
(100.0)
(1.1)
(14.4)
(4.8)
(3.4)
(8.2)
pNPP
ADP
AMP
Phosphoserine
Phosphothreonine
Glucose 6-phosphate
0.0
19.6
0.0
0.0
10.0
0.0
{
{
{
{
{
{
0.0
2.0
0.0
0.0
2.5
0.0
(0.0)
(5.5)
(0.0)
(0.0)
(2.8)
(0.0)
Note. Reaction conditions were as described under Materials and Methods. The concentration of all substrates was 1 mM. pNPP, pnitrophenol phosphate.
stimulate thylakoid activity, unlike that of vacuolar
PPase (cf. 4, 29). Furthermore, many divalent cations
substantially inhibit thylakoid PPase with a decreasing order of Cd2/, Ca2/, Co2/, Zn2/, Mn2/, and Cu2/.
Such an inhibitory effect is probably due to the competition of divalent cations to the active site of thylakoid
PPase. However, we cannot exclude the possibility that
they bind specifically to other domains of thylakoid
PPase and regulate its enzymatic activity. The actual
mechanism of the inhibition of these divalent cations
to thylakoid PPase is still not clear and deserves our
further investigation.
protein on thylakoid membrane as well as the purified
thylakoid PPase. Moreover, the serum containing antithylakoid PPase antibody inhibited the PPi hydrolysis
reactions of purified and membrane-bound PPases (Fig.
6). The antibody raised against vacuolar H/-PPase from
mung bean seedlings, kindly provided by Dr. Maeshima
(9), failed to recognize any components of thylakoid membrane and purified enzyme. Vice versa, the antibody
against purified thylakoid PPase did not cross-react with
that from mung bean vacuoles (data not shown). The
immunological studies indicate unequivocally that
PPases from mung bean tonoplast and spinach thylakoid
are structurally distinct proteins.
Immunological Studies
Antibody to the purified thylakoid PPase was raised
in a rabbit. As shown by the immunoblot analysis in Fig.
5, the serum reacted only with the component of 55 kD
Amino Acid Composition
The amino acid composition of thylakoid PPase is
shown in Table IV. The thylakoid PPase contains 507.6
TABLE III
Cation Requirements of Thylakoid Pyrophosphatase
Specific activity
Membrane-bound PPase
(mmol PPi /mg proteinrh (%))
Cations
None
Mg2/
Ca2/
Cd2/
Mn2/
Co2/
Cu2/
Zn2/
Li/
Na/
K/
Cs/
NH/
4
1.2
13.8
1.4
2.6
0.5
0.0
0.5
1.7
0.0
0.0
0.0
0.0
3.2
{
{
{
{
{
{
{
{
{
{
{
{
{
0.2
1.4
0.1
0.2
0.1
0.0
0.1
0.1
0.0
0.0
0.0
0.0
0.3
Purified PPase
(mmol PPi /mg proteinrh (%))
(8.7)
(100.0)
(10.1)
(18.8)
(3.6)
(0.0)
(3.6)
(1.2)
(0.0)
(0.0)
(0.0)
(0.0)
(2.3)
0.0
330.0
0.0
4.0
22.8
0.0
0.0
35.0
3.3
0.0
0.0
0.0
0.0
{
{
{
{
{
{
{
{
{
{
{
{
{
0.0
15.0
0.0
0.4
2.1
0.0
0.0
2.0
0.5
0.0
0.0
0.0
0.0
(0.0)
(100.0)
(0.0)
(1.2)
(6.9)
(0.0)
(0.0)
(10.6)
(1.0)
(0.0)
(0.0)
(0.0)
(0.0)
Note. Pyrophosphate activity was determined in the presence of 5.0 and 50 mM of divalent and monovalent cations (with Cl0 as counteranion), respectively, as described under Materials and Methods.
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JIANG ET AL.
FIG. 4. Effect of the concentration of Mg2/ on the thylakoid pyrophosphatase activity. The PPase activity was determined in the presence of Mg2/ at concentrations from 0 to 10 mM as indicated. The
concentration of PPi was 1 mM. Other reaction conditions and the
measurement of Pi release were as described under Materials and
Methods. The control enzymatic activity was approximately 350
mmol PPi consumed/mg proteinrh for purified PPases (l) and 10
mmol PPi consumed/mg proteinrh for thylakoid (s) PPases.
{ 17 amino acids/mol, resulting in a calculated mw of
54,746 { 1833, which is similar to that obtained by
gel filtration chromatography. The enzyme contains a
higher percentage of glutamate and glycine but limited
amounts of histidine, tyrosine, and cysteine. Thylakoid
FIG. 6. Effect of anti-pyrophosphatase antiserum on the activities
of membrane-bound (A) and purified (B) thylakoid PPases. (A) The
indicated amounts of anti-pyrophosphatase antiserum (l) and preimmune serum (s) were incubated with the thylakoid (40 mg) at
257C for 10 min, and then pyrophosphatase activity was measured.
The initial activity was approximately 8 mmol PPi consumed/mg proteinrh without antiserum. (B) Various amounts of anti-pyrophosphatase antiserum (l) and preimmune serum (s) were incubated with
2 mg purified thylakoid pyrophosphatase at 257C for 10 min, and
then the PPase activity was determined. The initial activity was
approximately 340 mmol PPi consumed/mg proteinrh without antiserum. The conditions for enzymatic reaction were as described under
Materials and Methods.
FIG. 5. Immunoblot analysis of specificity on anti-pyrophosphatase
antiserum. Lane 1, the standard markers with molecular masses
indicated on the left; lanes 2 and 3, gel stained with Coomassie
brilliant blue; lanes 4 and 5, immunoblot with anti-pyrophosphatase
antiserum and horseradish peroxidase-linked protein. Lanes 2 and
4, thylakoid preparation (25 mg); lanes 3 and 5, purified thylakoid
PPase (1 mg). The conditions for immunoblotting and visualization
were as described under Materials and Methods.
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PPase is slightly more negatively than positively
charged, 19.0 { 1.2% versus 10.5 { 0.6%. The content
of hydrophobic residues of thylakoid PPase is 57.1 {
2.8%. The amount of tryptophan was measured by the
method of Spandee and Witkop (17) as 13 { 3.6 tryptophan residues per mole of polypeptide, resulting in a
percentage of aromatic amino acid residues of 8.4 {
1.1%. The content of the cysteic acid is surprisingly
low, 4.7 { 2.1 residues/mol thylakoid PPase, partially
accounting for its low sensitivity to sulfhydryl group
inhibitors, such as NEM and NPM (see below).
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THYLAKOID PYROPHOSPHATASE
Inhibitor Sensitivity
TABLE V
Various chemical modifiers were utilized to probe
possible amino acid residues involved in thylakoid
PPase activity (Table V). Carboxylate modifiers DCCD
and EDC inhibited the PPase activity, but the inhibition could be protected by preincubation of PPase with
PPi and Mg2/. Moreover, thylakoid PPase was much
more sensitive to hydrophobic DCCD than its hydrophilic derivative EDC, indicating the essential carboxylate groups might reside in a more water-inaccessible
region. TNM, acetic anhydride, and DIDS could also
inhibit thylakoid PPase, with I50 values of 0.8, 7.5, and
7.5 mM, respectively. The inhibition by DIDS was substantially protected by substrate, while that by TNM
and acetic anhydride was partially prevented. Potent
modifiers of lysine residue, such as FITC, NBD-Cl, and
PLP, could not inhibit thylakoid PPase. Furthermore,
thylakoid PPase is insensitive to several other inhibitors of vacuolar H/-PPase such as maleiimides (30) and
PGO (31). We speculated that thylakoid PPase does not
contain essential cysteine or arginine residues in the
active site. The structure and the reaction mechanism
of these two membrane-bound PPases are expected to
be different. The difference may provide insights on the
architect of the proton channel and how the proton is
translocated across vacuolar membranes by PPases.
TABLE IV
Amino Acid Analysis of Thylakoid PPase
Amino acid
Number
Asp
Glu
Ser
Gly
His
Arg
Thr
Ala
Pro
Tyr
Val
Met
Cys
Ile
Leu
Phe
Lys
Trp
42.6
54.0
25.5
48.9
7.3
28.1
32.8
42.6
31.2
9.9
39.0
15.1
4.7
31.2
49.9
19.8
25.0
13.0
Total
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
2.1
1.1
0.6
1.1
0.6
0.6
0.6
0.0
0.6
0.6
1.1
1.1
2.1
0.6
0.0
0.0
0.6
3.6
507.6 { 17
%
8.2
10.4
4.9
9.8
1.4
5.4
6.3
8.2
6.0
1.9
7.5
2.9
0.9
6.0
9.6
3.8
4.8
2.5
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
{
0.4
0.2
0.1
0.2
0.1
0.1
0.1
0.0
0.1
0.1
0.2
0.2
0.4
0.1
0.0
0.0
0.1
0.7
100
Note. The procedures of amino acid analysis were described under
‘‘MATERIALS AND METHODS’’. The content of tryptophan was
determined by the method of Spandee and Witkop [17]. The apparent
molecular weight (Mapp) of PPase was measured by SDS-PAGE as
shown in Figure 4. The calculated molecular weight was determined
from amino acid analysis. Calculated Mr 54,746 { 1,833. Mapp 55,000.
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Inhibitor Sensitivities of Thylakoid PPase
I50 (mM)
Inhibitor a
Membranebound PPase
Purified
PPase
FITC
NBD-Cl
PLP
EDC
DCCD
NEM
NPM
DIDS
TNM
HNBB
NBS
Iodoacetate
Acetic anhydride
DEPC
PGO
BD
—
NE
—
NE
NE
NE
NE
NE
NE
—
—
—
5.0
—
—
—
NE
NE
NE
20.0 (//)
0.7 (//)
NE
NE
7.5 (//)
0.8 (/)
NE
NE
NE
7.5 (/)
NE
NE
NE
Note. Purified thylakoid PPase (2 mg) and thylakoid membrane (40
mg) were incubated with inhibitors for 20 min at room temperature.
After 20-fold dilution in reaction medium, the enzymatic activity was
assayed as described under Materials and Methods. I50 , determined
directly from each concentration curve, is the concentration of inhibitors at which the half-maximal inhibition was observed. The percentage of protection by substrate (Mg2/-PPi) is calculated as described
previously (30). NE, no effect (õ10%), —, not done; /, partially
protectable (Ç40%); //, fully protectable (ú75%).
a
Abbreviations used: BD, 2*,3*-butanedione; DCCD, N,N*-dicyclohexylcarbodiimide; DEPC, diethylpyrocarbonate; DIDS, 4-acetamido-4*-isothiocyanostilbene-2,2*-disulfonic acid; EDC, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide; FITC, fluorescein 5*-isothiocyanate; HNBB, 2-hydroxy 5-nitrobenzyl bromide; NBD-Cl,
7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; NBS, N-bromosuccinimide;
NEM, N-ethylmaleimide; NPM, N-phenylmaleimide; PGO, phenylglyoxal; PLP, pyridoxal 5*-phosphate; TNM, tetranitromethane.
The physiological function of thylakoid PPase is still
in dispute. Thylakoid PPase activity is independent of
photosynthetic electron transport and photophosphorylation (32). No positive evidence has indicated, so far,
that thylakoid PPase is associated with proton pumping (26, 33). A recent report indicated the presence of
inorganic pyrophosphate-dependent protein phosphorylation of thylakoid membrane (34). However, the PPimediated protein phosphorylation of thylakoid is lightand reducing agent-dependent. These properties exclude the possibility that thylakoid PPase is involved
in the protein phosphorylation of thylakoid membrane.
The elucidation of the exact role of thylakoid PPase
required further efforts. Nevertheless, the present
work is the first step toward understanding thylakoid
PPase of higher plants.
ACKNOWLEDGMENTS
This work was supported by a grant from National Science Council,
Taiwan, Republic of China (NSC86-2311-B007-012) to R.L.P. We sin-
arca
112
JIANG ET AL.
cerely appreciate Dr. M. Maeshima for the kind gift of anti-mung
bean vacuolar H/-PPase antibody.
18.
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