(Received for publication, August 26, 1994; and in revised form, December 27, 1994)
From the
Under unisite conditions (ratio of ATP to chloroplast coupling
factor (CFCF
), approximately 1:2.8), spinach
thylakoid ATPase depends on prior reductive activation of
CF
, just as multisite ATPase does, and is sensitive to
removal of CF
by EDTA. Faster rates in room light than in
semidarkness and up to 80% inhibition by uncouplers only in room light
indicate a strong effect of proton-motive force, which can be provided
by room light. In addition, unisite ATPase is inhibited by azide as
long as some ADP is bound to the CF
.
Several differences
were found between unisite and multisite ATPase. 1) The unisite
activities of both membrane-bound and free enzyme were stimulated up to
3-fold by 4 mM free MgCl (a strong inhibitor of
multisite ATPase). 2) Thylakoid unisite ATPase was inhibited by sulfite
(50% inhibition at 5 mM), a powerful activator of multisite
ATPase. This inhibition is attributed to a nonspecific ionic strength
effect. 3) Unisite ATPase was inhibited by trypsin treatment, which
increases multisite ATPase severalfold. 4) The pH profile of thylakoid
unisite ATPase is somewhat different from that of multisite. 5)
Alkylation of Cys-89 of the
subunit by N-ethylmaleimide
did not affect the unisite activity, but inhibited multisite activity
more than 90%.
Of the three catalytic sites of F-type ATPases, one was found to
have higher affinity for ATP than the
others(1, 2, 3, 4) . The properties
of this site have been studied extensively with both mitochondrial (1, 2, 5, 6) and bacterial
F-ATPases(3, 7) . The rate of ATP
hydrolysis when only one site is operating is between 10
and 10
times slower than with multisite operation.
There are also some different characteristics, for instance, a lower pH
optimum in both MF
(
)(6) and
EF
(7) .
With isolated F-ATPases
studied so far, azide is a strong inhibitor of multisite, but not of
unisite
ATPase(6, 7, 8, 9, 10) ,
and is therefore considered to be a specific inhibitor of cooperative
interactions between the catalytic sites. Lack of a major anion effect
on unisite activity appears to be another common property of MF
and EF
(11, 12, 13) .
However, a number of differences were also found. For instance,
millimolar free inorganic phosphate activated MF
unisite
ATPase 3-fold(14) , but slightly inhibited Escherichia coli unisite ATPase(7, 15) . Studies of chloroplast
unisite ATPase, including its kinetic properties, have been reported by
Gräber's group(4, 16) . We
were interested in exploring further biochemical parameters of
thylakoid unisite ATPase, to compare with unisite ATPase in other types
of F
, and with chloroplast multisite ATPase. This has
included the effects of different ions, uncouplers, trypsin treatment,
pH, energy transfer inhibitors, light, azide, and modification by
MalNEt. Our results indicate that chloroplast ATPase shares some
properties with other F-type ATPases, but it has some unique
characteristics at the unisite level. Also the characteristics of the
unisite catalysis differ significantly from those of multisite activity
of the same enzyme.
The reaction mixture
(60 µl) contained 20 mM Tricine-NaOH, pH 8.0, 10 mM DTT, 5 mM MgCl, and thylakoids at 30 µg
of Chl/ml. Other additions are as indicated under
``Results.'' Thylakoids were first added to the reaction
mixture without ATP, then 51 µl of this mixture were added to
another Eppendorf tube containing 9 µl of
[
-
P]ATP (100 nM, with specific
activity
10
cpm/nmol of ATP) to start the reaction,
with quick mixing by pipetting. For general activity assays, the
reaction was usually run for 60 s before addition of 25 µl of the
stop solution, 30 mM Hg(NO
)
in 5 M acetic acid. After centrifuging, 50 µl of the supernatant were
assayed for
P
(see below) released by unisite
ATPase. For kinetic assays, 90-µl timed aliquots were removed from
a larger volume of reaction medium and placed in an Eppendorf tube
containing 40 µl of stop solution.
To determine pH profiles, a
solution containing the buffers MES (pK 6.0), MOPS
(pK
7.1), Tricine (pK
7.9),
and CHES (pK
9.0) was mixed with Mg
and DTT prior to adjusting the pH. The final concentration of
each buffer was 20 mM, 5 mM Mg
, and
10 mM DTT.
For unisite ATPase, the reaction medium (50 µl) contained 50
mM Tricine-NaOH, pH 8.0, 10 mM DTT, with either 5
mM MgCl, or 8 mM CaCl
. The
ratio of ATP to CF
was 1:8 with 0.1 µM [
-
P]ATP and 0.8 µM CF
(0.33 mg protein/ml). The reaction was conducted at
room temperature for 60 s.
First, the formation of P by membrane-bound
enzyme was found to be dependent on prior reduction of the thylakoids (Fig. 1). ATP hydrolysis by the reduced enzyme was almost linear
up to 40 s at a turnover rate of 0.003 s
. By
comparison, enzyme activity of the nonreduced thylakoids was
insignificant, at most 5% of the rate and that for only the first 20 s.
Second, treating thylakoids with EDTA at low ionic strength removes
CF
and causes uncoupling reduced unisite activity more than
90% (data not shown). Third, preliminary experiments showed up to 50%
inhibition was found by addition of an antiserum against CF
(data not shown), but
10% inhibition was seen with the
preimmune serum. In these experiments the reduced thylakoids and
antiserum were incubated at 0 °C for 30 min and centrifuged, and
the thylakoids were resuspended and used for unisite ATPase assay.
Other conditions would have to be tested to see if greater inhibition
might be possible.
Figure 1:
Thylakoid unisite ATPase is activated
by DTT plus light reduction. The reaction medium (60 µl) contained
50 mM Tricine-NaOH, pH 8.0, 2 mM MgCl,
1.8 µg of Chl, and 15 nM [
-
P]ATP, with (for reduced thylakoids)
or without (for control thylakoids) 10 mM DTT. Thylakoids were
either untreated (hollow circles) or previously reduced by DTT
in the light (filled circles). The reactions were run in room
light (20 µE/m
s) at room temperature (22
°C).
Figure 2:
Gramicidin inhibits thylakoid unisite
ATPase in room light, but not in semidarkness. The reaction mixture (60
µl) contained 10 mM Tricine-NaOH (pH 8.0), 10 mM DTT, 5 mM MgCl, 1.8 µg of Chl, and 15
nM [
-
P]ATP. The reactions were run
for 60 s at room temperature, in either room light (20
µE/m
s; hollow circles) or semidarkness
(<2 µE/m
s; filled
circles).
Since it is known that
CF activation occurs with a relatively low
pmf(23, 24) , it seemed conceivable that even room
light may have generated enough electron transport for this purpose.
Accordingly the same experiments were carried out in very dim light
(<2 µE/m
s); and indeed, control rates were
much lower, and the uncoupler effect disappeared (Fig. 2).
Figure 3: Azide inhibits regular, but not ADP-depleted thylakoid unisite ATPase. The assay conditions were as in Fig. 2, in room light, with control (hollow circles) or ADP-depleted thylakoids (filled circles).
It was
shown previously that azide inhibits by enhancing the binding of
inhibitory MgADP (26, 27) to one of the three
catalytic sites in isolated thylakoids. To obtain ADP-depleted
membrane-bound ATPase, thylakoids were diluted in the light and washed
extensively with EDTA at high ionic strength right after illumination,
as described by others (4, 18) . Measurements of bound
adenylates showed a drop from 1.70 down to 0.76 nmol of bound ADP per
nmol of CF; and a drop of ATP from 1.77 to 0.95 nmol/nmol
of CF
. The depleted thylakoids showed almost no azide
inhibition of unisite ATPase (Fig. 3).
In our previous
studies, we found that venturicidin can specifically inhibit thylakoid
ATPase by blocking proton pumping through CF(17) .
Under unisite conditions, a maximum of 30-40% inhibition was
observed in a number of experiments.
Figure 4:
A, salts inhibit thylakoid unisite ATPase.
Assay conditions as in Fig. 2, in room light. The reactions were
run for 60 s. B, sulfite stimulates soluble CF unisite ATPase, but other salts have no effect. The reaction
mixture (60 µl) contained 50 mM Tricine-NaOH (pH 8.0), 10
mM DTT, 5 mM MgCl
, 100 nM [
-
P]ATP, 800 nM reduced
CF
, and different salts at different concentrations, as
indicated. The reactions were run for 60 s at room
temperature.
We conducted similar experiments with
purified soluble CF (Fig. 4B). Only sulfite
had an effect on the soluble enzyme activity, all other salts had no
effect. As expected sulfite stimulated the soluble unisite activity,
opposite to its effect on the thylakoid bound enzyme.
The inhibitory
effect of anions on thylakoid unisite ATPase can be observed only when
the reaction medium contains mM levels of free Mg (Fig. 5) and the assays are conducted in room light.
Without Mg
in the assay medium, or if the assay is
performed in very dim light (<2 µE/m
s), the
rates are much lower (see below), and added NaCl or
Na
SO
have either no effect or are slightly
stimulatory.
Figure 5:
Thylakoid unisite ATPase is inhibited by
salt only in the presence of Mg. The assay conditions
were similar to those in Fig. 2, in room light, except that
these assays were performed in either the presence (hollow
symbols) or absence (filled symbols) of 5 mM MgCl
. Open squares and filled
triangles, NaHSO
; open triangles and filled circles, NaCl.
Inorganic phosphate stimulates soluble
MF(14) , but inhibits EF
unisite
ATPase(6, 15) . With thylakoid unisite ATPase, less
than 1 mM phosphate enhanced the unisite ATPase activity, but
inhibited at concentrations above 1 mM (not shown). However,
the stimulatory effect of phosphate was found to be due to the
formation of ATP under room light (10-20
µE/m
s), presumably from the tightly bound ADP,
thereby raising the net ATP concentration. The inhibitory effect of
phosphate is probably due to the same anion inhibition found with other
salts. With soluble CF
, a slight stimulatory effect was
also observed under unisite conditions, but much less than that of
sulfite (data not shown).
Figure 6:
A, cation stimulation of thylakoid unisite
ATPase. The reaction mixture was similar to that in Fig. 2, in
room light, except for the presence of different salts (as indicated)
at the concentrations indicated. B, cation stimulation of
soluble CF unisite ATPase. The conditions were similar to
those in Fig. 2, with additions of CaCl
or
MgCl
as indicated.
More
surprisingly, a similar cation effect was also found with soluble
CF unisite ATPase (Fig. 6B), except that
here Ca
was more effective than Mg
,
in contrast to the thylakoid-bound enzyme. For instance, Mg
was optimal at 5 mM, giving a 2-fold stimulation; but
with Ca
, a 7-fold stimulation was observed at 10
mM.
We could think of two possibilities to explain
this result. The first is that trypsin digestion might destroy the high
affinity site, as the result of cleaving the and/or
subunits(30, 31) . If so, soluble CF
should also have been inhibited. However, trypsin digestion
increased soluble unisite activity only to some extent (data not
shown). Thus it is not likely that inhibition of thylakoid unisite
ATPase by trypsin is due to a loss of one or the other of the large
subunits. The second possibility, which is accordingly more likely, is
that the slight uncoupling effect of trypsin treatment (30) might cause the inhibition.
Figure 7:
The pH profiles of thylakoid ATPases. The
unisite (hollow circles) reaction medium (60 µl) contained
20 mM MES, 20 mM MOPS, 20 mM Tricine, 20
mM CHES, 5 mM MgCl, 10 mM DTT,
15 nM [
-
P]ATP, and 1.8 µg of
Chl. Multisite (filled circles) ATPase was run with 5 mM ATP, and other conditions were the same. The medium pH was
adjusted prior to the addition of ATP and thylakoids. The rates are
shown relative to each respective maximal rate. These were 0.002 mol of
P
/mol of CF
CF
for unisite
hydrolysis, and 28.5 for multisite.
Interestingly, very similar pH profiles
were also observed with soluble CF ATPase (data not shown).
Thus the differences between thylakoid unisite ATPase and other F-type
ATPases in the pH profiles are probably not due to the thylakoid
membranes, but rather to the reactivity of CF
itself.
Figure 8: Sulfite stimulates thylakoid unisite ATPase in semidarkness. The conditions were as in Fig. 2, in room light (hollow triangles) or semidarkness (closed circles). A, reduced thylakoids; B, thylakoids reduced and treated with trypsin.
Similar results were also obtained with trypsin-treated thylakoids. In dim light, sulfite stimulated the trypsin-cleaved thylakoid unisite ATPase activity (Fig. 8), with an optimal concentration at 20 mM, increasing the rate 3-fold. However, further increasing sulfite concentrations again resulted in some inhibition.
Data presented in this report demonstrate differences between
the unisite and multisite activities of thylakoid ATPase and between
chloroplast F and mitochondrial or E. coli F
- ATPases. These results could be useful for further
understanding the molecular mechanism of the enzyme.
First, we
wanted to make sure that the very low levels of ATP hydrolysis are due
to CFCF
. The dependence of unisite activity on
prior reduction of the thylakoids by DTT in the light, loss of activity
when thylakoids are depleted of CF
by EDTA treatment at low
ionic strength, and partial inhibition due to an antiserum against
CF
are all characteristics shared by normal thylakoid
multisite ATPase. It is highly improbable that another enzyme could be
responsive to these same inputs.
The rates of unisite ATPase we
observed in room light were comparable to those obtained in a similar
study by Fromme and Gräber(16) , despite
the differences in experimental conditions. They used either light or
an acid/base jump to reactivate the reduced thylakoid enzyme before
each assay, without comment on any activity in the absence of this
activation. They also used a high concentration of uncoupler (3-6
mM NHCl) in the assay medium after reactivating
the enzyme(16, 34) .
Our finding of inhibition of
unisite catalysis by uncouplers in room light but not in dim light
indicates a requirement for a small proton-motive force for all but a
small fraction of the unisite activity (Fig. 2, Table 1).
This could not have been due to carryover of some pmf from the previous
reductive activation, especially because uncoupler inhibition was found
to the same extent when the thylakoids had been reduced by incubating
with DTT in the dark, without PMS. Generation of a pmf under room light
(<25 µE/ms) and with no added
electron-carrying dye initially seemed unlikely. However, a very low pH
gradient, perhaps only 2.0 units, is sufficient to activate
CF
(23, 24) . With either 9-aminoacridine
or neutral red as indicators, we did observe a thylakoid pmf forming
due to these low light intensities and without added redox dyes. (
)The pattern of electron flow, in the absence of added
redox dyes, that can operate at this low light level has not been
defined. Preliminary experiments with inhibitors (dichlorophenyl
dimethylurea for PSII; high levels of KCN for plastocyanin and PSI)
seem to indicate involvement of only PSI; but further work is needed
for a better definition.
It was surprising to see that thylakoid
unisite ATPase (Fig. 3) and that of soluble CF are
partially inhibited by azide (Fig. 3). Both MF
and
EF
unisite activities, and even that of soluble CF
Ca
-dependent ATPase (9) , are not
inhibited by azide(6, 10) . The azide inhibition of
thylakoids (26, 35) as with other systems, is
considered to be due to tightening the binding of inhibitory MgADP to
one of the adenylate binding sites. Thereby it interferes with
site-site interactions and causes inhibition of steady state ATP
hydrolysis. This is likely to be the case with thylakoid unisite
activity also, since azide did not inhibit thylakoids which had been
partially depleted of bound ADP. Either the unisite binding and
hydrolysis occurs at the site of tight ADP binding, or ATP hydrolysis
at only one site is still subject to allosteric control by the tightly
bound ADP. Either way, any means of loosening or releasing the bound
ADP would stimulate unisite activity.
As observed with
MF(28) , venturicidin only partially inhibits
thylakoid unisite ATPase. Matsuno-Yagi and Hatefi (28) proposed
that venturicidin binding to the F
sector freezes its
structure, but does not block the proton channel. Since under unisite
conditions only one set of protons is expected to move through any one
channel, a slow translocation rate would be sufficient to support the
very slow ATP hydrolysis.
The effects of salts on thylakoid unisite
catalysis include stimulation at low concentrations (0-5
mM) and inhibition at higher levels. The inhibition is a
nonspecific anion effect (Fig. 4A) and is not found
with either unisite activity of soluble CF (Fig. 4B) or thylakoid multsite ATPase. Since it
does not occur in dim light, the salts may be interfering with either
the unknown electron flow path or in some other way with generation of
a pmf. While the cause is not known, the phenomenon explains why the
usually strong potentiation by sulfite cannot be observed at these
substrate levels.
Part of the stimulation by low levels of salts may
represent a modification of thylakoid structure. While the divalent
cations Ca and Mg
are most
effective, even Na
(Fig. 6A) and
Tricine (data not shown) have some effect. It was found much earlier (36, 37) that at very low salt concentrations the
grana structure of thylakoids disappears; but it can be restored by
adding salts, especially of divalent cations.
However, the greater
effectiveness of Mg, and especially the strong
stimulation of soluble CF
unisite ATPase by
divalent cations, indicates a specific role in either catalysis or
enzyme structure. This was quite unexpected; the usual concept is that
the role of divalent cations is only to complex with the adenylate,
forming the true substrate. In the present case the required
Mg
concentration of 4 to 5 mM is over 5
orders of magnitude higher than the substrate concentration (0.015
µM). It is even more strange in that free Mg
is a mild inhibitor of thylakoid multisite ATPase, but a severe
inhibitor for soluble CF
, with a K
of
10 µM(38, 39, 40) .
Guerrero et al.(41) proposed that free Mg inhibits ADP release from the tight binding site, thereby
stabilizing an inactive form of CF
. However, its failure to
inhibit the unisite activity seems at odds with that interpretation. It
seems likely that, on binding to CF
, Mg
induces a conformational change in favor of unisite binding and
hydrolysis of the substrate. A similar result was seen in MF
(42) where Mg
stimulated both unisite
catalysis and the rates of ADP and P
release from the
soluble enzyme.
Alkylation of Cys-89 in the light had little effect on unisite, but knocked out most of the multisite activity (Table 2), suggesting another difference between the two types of catalysis. Alkylation of Cys-89 may abolish site-site interactions(43) .
The minor component of unisite activity
found in very dim light was not inhibited by salts and (probably for
that technical reason) could show some stimulation by sulfite. Sulfite
also stimulates rather strongly the unisite activity of soluble
CF. In both cases, its action presumably has to do with
permitting release of the tightly bound, inhibitory MgADP, as in
multisite ATPase(41, 44) . Thus once again, with
CF
some site-site interactions, or at the least an
allosteric effect of ADP at an alternative site, are found under
unisite catalytic conditions.
Trypsin inhibition of thylakoid
unisite ATPase contrasts with the known stimulation of multisite ATPase
and also with its stimulation of the unisite activity by soluble
CF (data not shown). This inhibition is therefore most
probably attributed to its weak uncoupling of
thylakoids(30, 31) , since unisite catalysis cannot
renew the pmf during the reaction.
In conclusion, thylakoid unisite ATPase can be assayed under room light. Based on so many differences from multisite ATPase, it seems that the high affinity site is either a unique catalytic site or is one of the usual ones with very different characteristics, because of the very low ATP levels.
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