(Received for publication, August 7, 1995; and in revised form, September 11, 1995)
From the
Excess ATP is known to enhance Ca-ATPase
activity and, among other effects, to accelerate the Ca
binding reaction. In previous work, we studied the pH dependence
of this reaction and proposed a
3H
/2Ca
exchange at the
transport sites, in agreement with the
H
/Ca
counter transport. Here we
studied the effect of ADP and nonhydrolyzable ATP analogues on the
Ca
binding reaction at various pH values.
At pH 6,
where Ca binding is monophasic and slow, ADP,
adenosine 5`-(
,
-methylene)triphosphate (AMPPCP), or
adenyl-5`-yl imidodiphosphate (AMPPNP) increased the Ca
binding rate constant 20-fold. At pH 7 and 8, where
Ca
binding is biphasic, the nucleotides induce fast
and monophasic Ca
binding. At pH 7, AMPPCP
accelerated Ca
binding with an apparent dissociation
constant of 10 µM.
At acidic pH, ADP, AMPPCP, or AMPPNP
increased the equilibrium affinity of Ca for ATPase,
whereas at alkaline pH, these nucleotides had no effect. At pH 5.5,
AMPPCP increased equilibrium Ca
binding with an
apparent dissociation constant of 1 µM.
Ca-ATPase is a membranous enzyme which pumps
Ca
from the cytoplasm into the sarcoplasmic reticulum
lumen, requiring ATP hydrolysis. A simple description of the cycle is
given in Fig. S1. Cytoplasmic Ca
binding at
the transport sites is a crucial step in the ATPase cycle, as it
induces a change in the chemical reactivity of the catalytic site which
is either phosphorylatable by P
in the absence of
Ca
or phosphorylatable by ATP in the presence of
Ca
. Once the phosphoenzyme has been formed from ATP,
the Ca
bound at the transport sites can be released
into the lumen, and dephosphorylation occurs.
Figure S1: Scheme 1.
Ca-ATPase activity requires micromolar ATP and is
enhanced by submillimolar or millimolar
ATP(1, 2, 3, 4, 5) . As the
site of micromolar affinity is the catalytic site, there has been a
great deal of discussion as to whether the lower affinity site is the
catalytic or a regulatory site, especially because studies of ATP
binding at equilibrium have shown one or two different
sites(2, 5, 6, 7, 8) . ATP
is known to accelerate Ca
binding from the
cytoplasm(9, 10, 11, 12, 13) ,
Ca
release into the SR (
)lumen (14, 15) and
dephosphorylation(16, 17) . In other words, ATP
increases the rate of all steps which start from a nucleotide-deprived
species (steps 2, 3, and 4 in Fig. S1), suggesting that it binds
to the nucleotide site, even on the phosphoenzyme, so that ATP enters
the cycle as soon as ADP has dissociated. According to McIntosh and
Boyer(18) , Bishop et al.(19) , and Champeil et al.(16) , the regulation occurs on the
phosphoenzyme and probably at the catalytic site.
Cytoplasmic
Ca binding to the ATPase is particularly important in
studying the interaction between the catalytic and transport sites,
because it is the step at which ATPase changes its chemical
specificity. Keeping in mind that there is millimolar ATP in the
cytoplasm, understanding how ATP modulates the Ca
binding step will lead to a description of the Ca
binding mechanism closer to the in vivo phenomenon.
To analyze the effect of a nucleotide at the catalytic site on the
Ca binding reaction, the use of nonhydrolyzable ATP
analogues is more convenient than ATP, as it avoids perturbing the
Ca
binding reaction by the phosphorylation reaction.
ADP, AMPPCP, and AMPPNP have been used by different authors who reached
different conclusions. According to Ogawa et al.(20) , the Ca
affinity is increased by
AMPPCP; according to Fernandez-Belda et al.(21) ,
AMPPNP does not affect the affinity or the rate of
Ca
-binding; according to Wakabayashi and
Shigekawa(13) , AMPPNP increases the rate of Ca
binding.
These contradictory conclusions are probably due to
various experimental conditions, especially the pH. A previous study of
the Ca binding step has led us to a detailed
description of the 2Ca
/3H
exchange
occurring at the transport sites(22, 23) . Fig. S2describes this exchange without Mg
.
With Mg
, the species EH
and ECa
were shown to be phosphorylated by P
and ATP, respectively, so that Fig. S2can be taken as
step 4 in Fig. S1. On the basis of this previous work, we have
studied the effects of ATP on the cytoplasmic Ca
binding step at various pH values, using nonhydrolyzable ATP
analogues, and we show that ADP, AMPPNP, and AMPPCP have a pH-dependent
effect on both the rate and the affinity of Ca
binding. (i) These nucleotides drastically increase the rate of
Ca
binding at pH 6 whereas they have little effect at
pH 8. This is thought to occur via an increase in the rate of the
deprotonation steps which are rate-limiting without nucleotides. (ii)
These nucleotides also increase the Ca
affinity at
acidic pH, whereas they have no effect at neutral or alkaline pH. We
suggest this could be due to a higher affinity of the
Ca
-saturated ATPase for ATP analogues at acidic pH.
Figure S2: Scheme 2.
SR vesicles were prepared and tested as described in (22) from rabbits subjected to a 48-h starvation diet(24) . The experiments were carried out at 20 or 5 °C in thermostated rooms. Buffers were: 100 mM Tes-Tris (pH 8), 100 mM Mops-Tris (pH 7), or 100 mM Mes-Tris (pH 6.3-5.5) and were prepared with water filtered through a Milli-Q Water Purification System (Millipore). All salts were added as chlorides. FITC labeling was done as described in (16) . All nucleotides were purchased from Boehringer, ADP was the purest available preparation.
Equilibrium and kinetic measurements
involving Ca
were performed as described
in (23) . The rapid filtration apparatus (25) (Biologic, Chaix, France) was used to perfuse
Ca
for times from 50 ms to 5 s. Once SR
vesicles are adsorbed on the filter, the filter holder and the
perfusion device come into contact for the chosen perfusion time. The
perfusion of the buffer through the filter is performed by an
electronically controlled stepping motor. At the end of the perfusion,
the filter is removed by the manipulator.
The intrinsic fluorescence
change induced by the nucleotides was measured at equilibrium as
described in (5) , and the Ca binding
kinetics were measured using a stopped-flow apparatus (SFM3, Biologic,
Chaix, France) as described in (23) .
Free Ca concentrations were calculated as in (22) and using the
following figures for the dissociation constants of the calcium
complexes with nucleotides (in M): AMPPNP, 4
10
at pH 8, 5
10
at pH 7,
7
10
at pH 6; AMPPCP, 4
10
at pH 8, 4
10
at pH 7,
8
10
at pH 6.3, 1.3
10
at pH 6.
Figure 1:
Effect of nucleotides on Ca binding kinetics at pH 6 and 8. Ca
binding was
monitored at 20 °C by intrinsic fluorescence in the presence of 100
and 10 µM free Ca
, at pH 6 and 8,
respectively, without added Mg
. When specified, 0.1
mM nucleotide was present in both syringes. 1,
control; 2, ADP; 3, AMPPCP; 4,
AMPPNP.
Similar experiments were performed by measuring
radioactive Ca binding by the rapid filtration
technique, which allows evaluation of the Ca
specifically bound to ATPase. At pH 6 where Ca
binding was monophasic and slow, AMPPCP as well as AMPPNP
increased the Ca
binding rate (Fig. 2), thus
confirming the results obtained from intrinsic fluorescence. Increasing
the nucleotide concentration from 0.1 to 0.3 mM did not change
the kinetics. The kinetics were identical, whether or not ATPase was
incubated with the nucleotide, indicating that the binding of the
nucleotide is faster than Ca
binding. At pH 8, it was
very difficult to measure a clear acceleration of Ca
binding in the presence of AMPPCP or AMPPNP, due to the small
amplitude of the slow phase. Therefore, another series of experiments
was performed at pH 7 and 5 °C, varying the concentration of AMPPCP (Fig. 3). Under these conditions and without AMPPCP,
Ca
binding was biphasic. The fast phase, which
represented half of the stoichiometry, was too fast to be measured by
the filtration technique, even in absence of AMPPCP. The slow phase was
accelerated with an apparent dissociation constant of 10 µM (see inset in Fig. 3).
Figure 2:
Effect of AMPPCP on Ca
incorporation kinetics at pH 6.
Conditions were 3 mM Mg
, 30 µM free Ca
, 20 °C. Open symbols,
control; closed symbols, 0.1 mM AMPPCP was perfused
together with
Ca
.
Figure 3:
Effect of AMPPCP on Ca
incorporation kinetics at pH 7.
Conditions were 1 mM Mg
, 18 µM free Ca
, 5 °C. When present, AMPPCP was
perfused together with
Ca
.
, no
nucleotide;
, 3 µM;
, 10 µM;
, 30 µM;
, 100 µM. Inset, slow phase rate constant as a function of
AMPPCP.
Similar experiments were
performed using FITC-labeled SR. In FITC-labeled SR, ATPase has
covalently bound one molecule of FITC at Lys-515 which impairs
nucleotide binding at the catalytic site (28, 29, 30, 31) . The results
obtained at pH 6 and with 3 mM Mg are shown
in Fig. 4. With native SR, 0.1 mM AMPPCP increased the
binding rate constant for free Ca
at 30 µM from 1.5 to 7.4 s
, whereas with FITC-labeled
SR, AMPPCP did not affect Ca
binding kinetics which
had a rate constant of 2.5 s
. Similar results were
obtained when the nucleotide was AMPPNP without added
Mg
. Note that with FITC present at the catalytic
site, the Ca
binding kinetics were faster than
without FITC. This, together with the absence of effect of the
nucleotide with FITC-labeled ATPase shows that acceleration of the
Ca
binding kinetics is mediated by the nucleotide at
the catalytic site.
Figure 4:
Effect of AMPPCP on Ca
incorporation kinetics at pH 6 by
FITC-labeled SR. Conditions were 3 mM Mg
, 30
µM free Ca
, 20 °C. Open
symbols, control; closed symbols, 0.1 mM AMPPCP
was perfused together with
Ca
.
Figure 5:
Effect of nucleotides on Ca binding at equilibrium in presence of 3 mM Mg
. Empty symbols, no nucleotide; closed symbols, 0.3 mM nucleotide was perfused
together with
Ca
.
, no nucleotide;
, AMPPCP;
, AMPPNP;
, ADP. Continuous lines are best fits using the Hill equation. Denoting by Ca
the level of Ca
bound to ATPase and by Ca the
free Ca
concentration, the Hill equation, i.e. Ca
= Ca
/(1 +
(Ca
/Ca)
), allows
evaluating Ca
, the stoichiometry; Ca
, the
half-saturation concentration; and n
, the Hill
coefficient of the Ca
binding
curve.
The experiments done with Mg and ADP or AMPPNP
displayed an additional Ca
binding stoichiometry
which proved to be due to slow Ca
transport (data not
shown). This led us to check the purity of the nucleotides by HPLC,
using an ion exchange column (TSK DEAE-2SW). AMPPCP and ADP showed a
single peak, whereas AMPPNP was not pure. The additional stoichiometry
with ADP is thus more likely due to ATP synthesized by myokinase which
contaminates the SR preparation(24) . The impurities in AMPPNP
could possibly induce Ca
transport.
Fig. 6summarizes the effects of all three nucleotides,
showing that they decreased Ca at acidic pH, with or
without added Mg
. Note the specific effect of AMPPNP
in the presence of 3 mM Mg
which increased
the Ca
affinity at all pH values.
Figure 6:
Effect of pH and nucleotides on
Ca. Evaluation of Ca
was obtained as
explained in the Fig. 5legend.
, no nucleotide;
,
AMPPCP;
, AMPPNP;
, ADP. A, no
Mg
; B, 3 mM
Mg
.
The experiments
described above show that ADP and ATP analogues undoubtedly bind to the
various forms of ATPase which participate in the Ca binding reaction and that the binding of these nucleotides at
acidic pH results in an increase in the affinity for
Ca
. The next step was thus the evaluation of the
affinity of this nucleotide site.
The apparent affinity for AMPPCP
was measured at equilibrium and at various pH values. These experiments
are shown in Fig. 7. They were conducted by measuring the
Ca bound to ATPase at various AMPPCP concentrations,
keeping the free Ca
concentration fixed at a value
close to Ca
. For example, at pH 6 and without added
Mg
, the free Ca
concentration was
fixed at 6 µM which gives half-saturation. Addition of
AMPPCP induced an increase in the amount of bound Ca
up to saturation. This curve thus reflects the increase in the
affinity for Ca
due to AMPPCP addition. Its shape is
that of noncooperative binding curve, corresponding to the binding of
AMPPCP at a single site with an apparent dissociation constant of 10
µM.
Figure 7:
Effect of AMPPCP on Ca
incorporation at equilibrium and at
various pH.
, pH 7, 0.4 µM Ca
;
, pH 6.3, 3 µM Ca
;
, pH
6.3, 3 mM Mg
, 6 µM Ca
;
, pH 6, 6 µM Ca
;
, pH 5.5, 50 µM Ca
. Continuous lines are best fits
using the Michaelis-Menten equation.
This experiment was repeated at various pH values,
with and without Mg (Fig. 7). The curve was
flat at pH 7, as expected from the absence of effect of 0.3 mM
AMPPCP under similar conditions in Fig. 6. The curve was
sigmoidal at pH 5.5, (
)and the corresponding apparent
dissociation constant was 1 µM. The effect of
Mg
is illustrated at pH 6.3, where the apparent
dissociation constant for AMPPCP was 15 µM without
Mg
and 1 µM with 3 mM
Mg
.
To analyze the pH sensitivity of the
nucleotide affinity which modifies the Ca binding
reaction, we also needed to determine whether the nucleotide affinity
was pH-sensitive in the absence of Ca
. These
measurements were monitored by measuring the intrinsic fluorescence
response to nucleotide addition. They are summarized in Fig. 8,
which shows N
, the nucleotide concentration
yielding half-saturation of the fluorescence signal, as a function of
pH. As already shown for ATP(5) , neither of the two ATP
analogues we used was found to have a pH-sensitive affinity in the
absence of Ca
. Note that the affinities of these
nucleotides are close to that of ATP and that ADP is the only
nucleotide displaying a pH-sensitive affinity in the absence of
Ca
and Mg
.
Figure 8:
pH dependence of N. N
is the nucleotide concentration inducing half
of the intrinsic fluorescence maximal variation induced by nucleotide
addition in the presence of EGTA (0.4 mM at pH 8, 1 mM at pH 7, and 4 mM at pH 6 and 5.5).
, AMPPCP;
, AMPPNP;
, ATP;
, ADP.
In studying the effects of ADP and ATP analogues on the
Ca binding reaction, we have found that (i) ADP,
AMPPCP, and AMPPNP increased the rate of Ca
binding
10-30-fold at pH 6 and to a smaller extent at pH 7 and 8 (Fig. 1Fig. 2Fig. 3), (ii) FITC labeling induced a
2-fold increase in the Ca
binding rate at pH 6, and
nucleotides had no additional effect (Fig. 4), (iii) ADP,
AMPPCP, and AMPPNP increased the Ca
affinity at
acidic pH, whereas they had no effect at neutral or alkaline pH, except
for AMPPNP in the presence of Mg
(Fig. 5Fig. 6Fig. 7), (
)(iv) the
affinities of ATP, AMPPCP, and AMPPNP for Ca
-deprived
ATPase were not sensitive to pH, at variance with that of ADP (Fig. 8).
A few papers have reported the existence of a
pH-dependent equilibrium between Ca-deprived forms of
ATPase and suggest that this explains the inhibition of Ca
binding by protons at
equilibrium(22, 32, 33, 34) . The
Ca
binding kinetics are also pH-dependent, and this
is illustrated by Fig. S2, where the
Ca
-deprived ATPase exists under different protonated
forms. At pH 6, where Ca
binding is slow, this model
predicts that all ATPase is in the EH
form. At pH
8, where Ca
binding is biphasic, half the ATPase is
in the EH form and the other in the E form, and, at
pH 9, where Ca
binding is fast, all ATPase is in the E form. Thus, the deprotonation steps are thought to be
rate-limiting, probably because they induce some slow conformational
changes which were not explicitly described in the model (22, 23) .
Because at alkaline pH, Ca binding is fast and has a high affinity, the Ca
binding acceleration effect of nucleotides at acidic pH could be
explained by a nucleotide-induced deprotonation of the
Ca
-deprived ATPase. That is, if at pH 6 and in
presence of a nucleotide the predominant form was E instead of EH
in Fig. S2, this would increase both the
Ca
binding rate and affinity.
As reported above, we observed
that ADP also accelerates Ca binding, but at variance
with the triphosphate analogues, ADP displayed a higher affinity for
Ca
-deprived ATPase at pH 8 than at pH 6 (Fig. 8), and this could possibly deprotonate ATPase at pH 6.
This assumption was checked using the previously measured pK values of Ca
-deprived ATPase(22) . Given
these, the ADP affinity should be much more sensitive to pH to induce a
significant change in the distribution of the
Ca
-deprived ATPase. Thus, according to our model,
even in the case of ADP, the changes induced in the affinity are too
small to cause deprotonation. We can thus conclude that the
pH-dependent equilibrium of Ca
-deprived ATPase is not
modified by the presence of ADP, ATP, AMPPNP, or AMPPCP.
Scofano et al.(9) ,
Guillain et al.(10) , Fernandez-Belda et
al.(12) , and Stahl and Jencks (11) reported that
ATP increases the rate of the slow conformational change occurring
during the Ca binding reaction. We show here that
this increase is pH-dependent and is related to the deprotonation steps
occurring during Ca
binding. In addition, it is
controlled by the catalytic site and not restricted to ATP nor to the
fact that the nucleotides are hydrolyzable as suggested by
Fernandez-Belda et al.(21) . It is rather an effect of
adenine di- or triphosphate bound at the catalytic site, and it does
not require the presence of Mg
.
Figure S3: Scheme 3.
The first
assumption relies on the fact that ATP has a pK of 6.5 and ADP
of 6.4, so that the HATP or HADP complexes appear in the right pH
range. For the ATP analogues, the values of the pK are not
known. Nevertheless, we measured the dissociation constants of the
calcium-nucleotide complexes at various pH and compared them with that
of the CaATP complex. From this comparison, it appears that AMPPCP has,
as ATP, a dissociation constant sensitive to pH between 6 and 8.
However, the CaAMPPNP dissociation constant was found almost
insensitive to pH, so that nucleotide protonation should not be
responsible for the increase in Ca affinity.
The
second assumption thus seems more likely. If there were a species such
as EHCa (Fig. S3) with a pK of 6
and a higher affinity for the nucleotide than all the other species, it
would almost certainly not contribute to Ca
binding
in the pH 8 to 6 range in absence of a nucleotide. However, with a
nucleotide and below pH 7, it would increase the affinity for
Ca
and thus induce the increase in bound
Ca
seen in Fig. 7.