(Received for publication, November 7, 1994)
From the
In comparative experiments with Ca ATPase in
native sarcoplasmic reticulum vesicles and reconstituted
proteoliposomes, we find that a variable stoichiometry of
Ca
or Sr
transport per ATPase cycle
is observed in the absence of passive leak through independent
channels. The observed ratio is commonly lower than the optimal value
of 2 and depends on the composition of the reaction mixture. In all
cases, a progressive rise in the lumenal concentration of
Ca
and Sr
is accompanied by a
parallel reduction of coupling ratios. Significant ATPase activity
remains even after asymptotic levels of Ca
accumulation are reached. This residual activity subsides if the
Ca
concentration in the outer medium is reduced below
activating levels (as it would following Ca
transients in muscle fibers). The reduction of stoichiometric
coupling is explained with a reaction scheme, including a branched
pathway for hydrolytic cleavage of phosphorylated intermediate before
release of Ca
into the lumen of the vesicles. Flux
through this pathway is favored when net lumenal Ca
dissociation from the phosphoenzyme is impeded and results in
P
production accompanied by lumenal and medium
Ca
exchange. Occurrence of reactions through branched
pathways may have general implications for the stoichiometric
efficiency of energy-transducing enzymes.
Vesicular fragments of sarcoplasmic reticulum (SR) ()membrane provide a convenient experimental system for
studies of active Ca
transport coupled to ATP
utilization. Early experiments with this system (Hasselbach, 1964)
indicate that two Ca
are transported into the lumen
of the vesicles in parallel with utilization of one ATP. It was later
demonstrated that each ATPase requires cooperative binding of two
Ca
for catalytic activation (Meissner, 1973; Inesi et al., 1980). Therefore, transport of 2
Ca
/catalytic cycle occurs if both bound
Ca
are translocated and is equivalent to maximal
stoichiometric efficiency of the pump.
Ratios of 2 (or nearly 2)
Ca/ATP utilized have been observed under conditions
permitting free Ca
to remain low in the lumen of the
vesicles: (a) in steady state experiments in which oxalate is
used for complexation of lumenal Ca
(Martonosi and
Feretos, 1964) and (b) in pre-steady state experiments in
which lumenal Ca
has yet to rise (Inesi et
al., 1978). On the other hand, Ca
/ATP ratios
lower than 2 are commonly observed under other conditions permitting
lumenal Ca
to rise. Such lower ratios have been
generally attributed to leak of transported Ca
through pathways other than the ATPase. In fact, most diagrams
represent the ATPase cycle with a pathway of sequential reactions
which, in principle, do not allow uncoupling of Ca
transport and ATP utilization (Fig. 1, solid
line). On the other hand, a variable stoichiometry of active
transport may be considered to be an intrinsic feature of the pump if
the reaction scheme includes alternate pathways leading to hydrolytic
cleavage of P
without vectorial displacement of
Ca
(Johnson et al., 1985; Berman and King,
1990). We have now obtained experimental evidence for this latter
alternative, using native SR and reconstituted vesicles to measure
coupling ratios in the presence of gradually rising lumenal
Ca
(or Sr
) and in the virtual
absence of passive leak. We find that the optimal 2:1 ratio is reduced
in proportion to the lumenal concentration of transported cation. The
uncoupling is accounted for by a branched pathway (Fig. 1, dotted line) of the ATPase cycle, permitting cleavage of the
phosphorylated intermediate before net release of Ca
into the lumen of the vesicles.
Figure 1:
A simple diagram of the Ca ATPase catalytic cycle. Only four measurable reactions are shown
here: Ca
binding to the enzyme on the outer surface
of the vesicles in exchange for H
, enzyme
phosphorylation by ATP, dissociation of bound Ca
in
the lumen of the vesicles, and hydrolytic cleavage of the
phosphorylated enzyme intermediate. Isomeric transitions of these
intermediates (such as E1 to E2, and E-P1 to E-P2) are required to explain vectorial translocation of bound
Ca
(De Meis and Vianna, 1979) and the kinetic
behavior of the enzyme in transient state (Froelich and Taylor, 1975;
Inesi et al., 1980; Petithory and Jencks,
1986).
Octaethylene glycol-n-dodecyl ether
(CE
) was obtained from Nikko Chemical Co
(Tokyo). Purified egg yolk phosphatidylcholine and phosphatidic acid
were from Avanti Polar Lipid Inc.,
Ca
and [
-
P]ATP were from DuPont NEN, n-octyl-
-D-glucopyranoside, calcimycin (A23187),
and all other materials were obtained from Sigma.
SR vesicles were
prepared from rabbit hind leg muscle by the method described by Eletr
and Inesi(1972). This method selects vesicles derived from longitudinal
SR membrane and containing negligible amounts of the ryanodine receptor
Ca channel associated with junctional SR. The protein
concentration was measured by the method of Lowry et
al.(1951), standardized with bovine serum albumin.
Preparation
of unilamellar liposomes by reverse phase evaporation and
reconstitution of Ca transport ATPase with liposomes
were carried out as described previously (Yu et al., 1994).
The size, the ATPase orientation, and the low permeability of these
proteoliposomes were recently characterized in detail (Levy et
al., 1992).
Ca uptake by native SR vesicles
was followed by determining the association of radioactive
Ca tracer with the vesicles after elimination of the
reaction medium by filtration through Millipore 0.45-µm HWP
nitrocellulose membrane. The reaction mixture contained 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO
, 0.2 mM (
Ca)CaCl
, 0.2 mM EGTA, and 20
µg of SR protein/ml. The reaction was started by the addition of
0.1 mM ATP.
Measurements of lumenal and medium
Ca exchange were obtained by first loading the SR
vesicles with
Ca
as above for 3 min and
then adding 4 ml of reaction medium containing non-radioactive
Ca
to 1 ml of reaction mixture. The reaction was
stopped at serial times by addition of 3 ml of LaCl
solution (4 mM LaCl
, 10 mM PIPES,
pH 7.0). The quenched samples were then filtered through Millipore
membranes and washed with 10 ml of LaCl
, 10 mM PIPES, pH 7.0. The radioactivity was determined by scintillation
counting.
Ca or Sr
uptake by the
proteoliposomes was measured by continuously monitoring differential
(750/650 nm) absorption changes undergone by the metallochromic
indicator Arsenazo III (Scarpa, 1979). The reaction was carried out in
the cuvette of an SLM-Aminco double wavelength spectrophotometer. In
most cases the reaction medium contained 10 mM PIPES, pH 7.0,
100 mM KSCN (or K
SO
), 5 mM MgSO
, 50 µM Arsenazo III, and 0.8 mg of
proteoliposomal lipid/ml. CaCl
or SrCl
was
added to the cuvette after obtaining the absorption base line, and the
reaction was started by addition of 0.1 mM ATP. The output
from the spectrophotometer was recorded directly or acquired with a
Nicolet digital oscilloscope.
ATP hydrolysis in native vesicles was
followed by measuring production of P by the calorimetric
method described by Lanzetta et al.(1979). The reaction
mixture contained 10 mM PIPES, pH 7.0, 100 mM KCl, 5
mM MgSO
, 0.2 mM CaCl
, 0.2
mM EGTA, and 20 µg of SR protein/ml. The reaction was
started by the addition of 0.1 mM ATP and quenched at serial
times by mixing an aliquot with the color reagent.
ATP hydrolysis by
reconstituted proteoliposomes was measured by enzyme coupled assay
according to the method described by Horgon et al.(1972). The
absorption change at 340 nm was continuously monitored in a mixture
containing 10 mM PIPES, pH 7.2, 100 mM KCl, 5 mM MgSO, 20 µM CaCl
, 400 µg
of proteoliposomal lipid/ml, 25 units of pyruvate kinase/ml, 25 units
of lactic dehydrogenase/ml, 2 mM phosho(enol)pyruvate, and 150
mM NADH. The reaction was started by addition of 0.1 mM of ATP.
Phosphorylated intermediate formation by the
Ca ATPase of native SR vesicles was determined by
measuring the incorporation of [
-
P]ATP
terminal phosphate into the protein. The reaction mixture contained 10
mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO
, 0.2 mM EGTA, 0.2 mM CaCl
, and 20 µg of SR protein/ml. The reaction was
started by the addition of 0.1 mM [
-
P]ATP and quenched at serial time by
mixing aliquots of the mixture with equal volumes of 1 M perchloric acid and 4 mM NaH
PO
.
The denatured protein was collected on 0.45-µm Millipore filters
and washed with 30 ml of 0.125 M perchloric acid and 2 mM NaH
PO
. The filters were then dissolved in N,N-dimethylformamide and the radioactivity determined by
scintillation counting.
Figure 2:
Ca uptake and ATP
hydrolysis by native SR vesicles in the presence of oxalate. Reaction
mixture: 20 µg of SR protein/ml, 10 mM PIPES, pH 7.0, 100
mM KCl, 5 mM MgSO
, 0.2 mM (
Ca)CaCl
, 0.2 mM EGTA, and 5
mM oxalate. The reaction was started by the addition of 0.1
mM ATP. 25 °C. The P
release shown is the
difference between P
release in the presence and in the
absence of Ca
.
If similar
experiments are repeated in the absence of oxalate, the ATP-dependent
Ca accumulation soon reaches an asymptotic level, as
the concentration of lumenal Ca
rises rapidly (Fig. 3A). The ATPase activity, however, is not
inhibited in parallel with accumulation of Ca
.
Therefore, the Ca
:ATP ratio is reduced within a few
seconds after the addition of ATP. This phenomenon can be observed with
a better time resolution at low temperature (Fig. 3B),
due to the lower rates of catalytic and transport activity. The initial
Ca
:ATP ratio of nearly two demonstrates that the
native functional state of the enzyme is retained in these
preparations.
Figure 3:
Ca uptake and ATP
hydrolysis by native SR vesicles in the absence of oxalate. Reaction
mixture: 20 µg of SR protein/ml, 10 mM PIPES, pH 7.0, 100
mM KCl, 5 mM MgSO
, 0.2 mM CaCl
, and 0.2 mM EGTA. The reaction was
started by the addition of 0.1 mM ATP. A, 25 °C;
1.0 mM EGTA was added where indicated. B, 10
°C.
It is noteworthy that the decline in Ca accumulation is not due to passive leak through collateral
pathways since, if the ATPase activity is stopped by addition of the
highly specific inhibitor thapsigargin (Sagara and Inesi, 1991), no
significant leak of the accumulated Ca
is observed (Fig. 4). Notwithstanding the lack of net loss of Ca
load, lumenal and medium Ca
undergo rapid
exchange which is also prevented by thapsigargin (Fig. 5, see
also Takakuwa and Kanazawa (1981) and Soler et al.(1990)). It
should be pointed out that very little ADP-ATP or P
-ATP
exchange (i.e. energy conserving reversal of the cycle through
the solid line in Fig. 1, see also Ebashi and Lippman(1962); De
Meis and Carvalho, 1974) occurs under our experimental conditions,
owing to the very low ADP and P
concentrations. On the
contrary, Ca
exchange is accompanied by net P
production.
Figure 4:
Ca accumulated by native
SR vesicles in the absence of oxalate is not released following
addition of thapsigargin (TG). Ca
uptake was
obtained as described for Fig. 2at 25 °C. Thapsigargin was
added where 30 s following ATP, to reach a 1.0 µM concentration which inhibits the ATPase
completely.
Figure 5:
Ca exchange by loaded
native SR vesicles. Reaction mixture: 20 µg of SR protein/ml, 10
mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO
, 0.2 mM (
Ca)CaCl
, 0.2 mM EGTA. The
reaction was started by the addition of 0.1 mM ATP. 4 volumes
of reaction medium with non-radioactive Ca
was added
at 3 min; 25 °C.
The comparative experiments performed in the
presence and in the absence of oxalate indicate that the stoichiometric
ratio of Ca transport and ATP utilization is reduced
by the Ca
concentration rise in the lumen of the
vesicles. Furthermore, they indicate that high lumenal Ca
favors hydrolytic cleavage of intermediate through an alternate
pathway which results in production of P
and exchange of
lumenal and medium Ca
, but no net release of
Ca
into the lumen of the vesicles (pathway indicated
by a dotted line in Fig. 1).
Figure 6:
Phosphoenzyme levels in SR vesicles with
high or low lumenal Ca. Reaction mixture: 20 µg
of SR protein/ml, 10 mM PIPES, pH 7.0, 100 mM KCl, 5
mM MgSO
, 0.2 mM MgCl
, and 0.2
mM EGTA in the presence or absence of 5 µM A23187. The reaction was started by the addition of 0.1 mM [
-
P]ATP. Incubation in
ice.
Figure 7:
Ca (A) or
Sr
(B) uptake and ATPase activity sustained
by reconstituted proteoliposomes. Reaction mixture: 0.4 mg of
proteoliposomal lipid/ml, 10 mM PIPES, pH 7.0, 100 mM KSCN, 5 mM MgSO
, and 20 µM Ca
or 100 µM Sr
.
The mixture for Ca
uptake contained Arsenazo III, and
that for ATPase containing a coupled detection system as described
under ``Materials and Methods.'' The reaction was started by
the addition of 0.1 mM ATP. Thapsigargin was added 10 min
following the addition of ATP to reach a 1.0 µM concentration. 25 °C.
Transport inhibition by lumenal Ca or
Sr
can be attributed to saturation of the
phosphoenzyme Ca
binding sites in their state of low
affinity and inward orientation, with consequent accumulation of loaded
intermediate (E-PCa
in Fig. 1). It is of
interest in this regard that acid pH lowers, and alkaline pH increases
the affinity of the Ca
sites in both the outward and
inward orientations (Verjovski-Almeida and De Meis, 1977). In this
connection, we reported previously (Yu et al., 1993) that
higher levels of Ca
accumulation are obtained when
isothiocyanate, rather than chloride or sulfate, is the prevalent
anion, due to the ability of isothiocyanate to dissociate H
and compensate for lumenal H
loss consequent to
H
/Ca
countertransport. We then
performed experiments to check whether replacement of isothiocyanate
with sulfate would influence net Sr
uptake and
Sr
/ATP stoichiometry by affecting the affinity of the
inward oriented Ca
sites. In fact, we found that when
sulfate rather than isothiocyanate was used, both the asymptotic level
of Sr
uptake and the Sr
/ATP ratio
were reduced (Fig. 8).
Figure 8:
Sr uptake by
reconstituted proteoliposomes in the presence of different anions.
Reaction mixture: 0.4 mg of proteoliposomal lipid/ml, 10 mM PIPES, pH 7.0, 100 mM K
SO
(A) or KSCN (B), 5 mM MgSO
, and 100 µM Sr
.
The reaction were started by the addition of 0.1 mM ATP; 25
°C.
The comparative experiments on
Sr and Ca
uptake, as well as on the
effects of sulfate and isothiocyanate, indicate that inhibition of net
Ca
uptake and reduction of stoichiometric efficiency
are both related to the extent of divalent cation binding to the enzyme
sites exposed to the lumen of the vesicles. In this context, the
stoichiometric efficiency of the pump is a kinetically controlled
variable which decreases progressively as the lumenal concentration of
transported cation rises. It should be pointed out that in no instance
we found the Ca
/ATP ratio to exceed the value of two,
which is the maximal ratio permitted by the stoichiometry of enzyme
sites.
The idea of uncoupling in energy transducing enzymes derives
from the effect of ionophores such as paranitrophenol and carbonyl
cyanide p-trifluoromethoxyphenylhydrazone which facilitate
leak of H across mitochondrial membranes, thereby
collapsing the electrochemical gradient and uncoupling electron
transport from ATP synthesis (Mitchell, 1979; Penefsky and Cross,
1991). An analogous effect is produced by ionophores such as X-537A or
A23187 which produce leak of Ca
across SR membranes,
thereby preventing formation of transmembrane Ca
gradients and uncoupling ATP utilization from net Ca
accumulation (Scarpa et al., 1972). On the other hand,
we find that the stoichiometric ratio of Ca
uptake
and ATPase activity can be also reduced in the absence of passive
Ca
leak. Therefore, the phenomenon observed in these
experiments is inherent to the ATPase mechanism itself. We also find
that, given an initial coupling ratio for native SR or proteoliposomal
vesicles (which may be influenced by the reaction mixture and/or the
enzyme preparation), such a ratio is reduced in parallel with rise of
lumenal Ca
(or Sr
) and saturation
of the lumenal phosphoenzyme sites with the divalent cation. While net
Ca
uptake is reduced by the rise of lumenal
Ca
, the ATPase activity is inhibited most effectively
by a reduction of the Ca
concentration in the outer
medium rather than by the rise of lumenal Ca
.
It
is apparent from the reaction scheme in Fig. 1(solid lines only) that a rise of lumenal Ca ([Ca
]
) would interfere
with progress of the E-P
Ca
intermediate
through the cycle, thereby producing inhibition of both Ca
uptake and ATPase activity with no change of their coupling
ratio. Therefore, in order to explain the observed uncoupling effect of
lumenal Ca
in the absence of a Ca
leak, it is necessary to postulate an alternate pathway for
hydrolytic cleavage of the intermediate before release of bound
Ca
into the lumen of the vesicles (dotted lines in Fig. 1). The occurrence of lumenal and medium
Ca
exchange, in the absence of energy conserving
ATP-ADP and P
-ATP exchanges, is consistent with significant
flux through this alternate pathway (dotted line in Fig. 1). Furthermore, the increased steady state level of
phosphorylated intermediate observed in the presence of high lumenal
Ca
suggests that the rate constant of the alternate
pathway is lower than that for the primary pathway.
We tested these
intuitive predictions by adding an alternate pathway to a reaction
sequence previously utilized to calculate steady state fluxes and
levels of intermediates, based on the bidirectional rate constants of
the partial reactions and the concentrations of substrates and
products. The reaction sequence (Fig. 9) includes second order
association and dissociation events, as well as isomeric transitions as
required to explain the vectorial changes of orientation, and the
kinetic and cooperative behavior of the enzyme under different
conditions (Inesi et al., 1980; Fernandez-Belda et
al., 1984). With specific reference to the branched pathway, we
can see in the diagram (Fig. 9) that the intermediate E`-PCaCa, formed by phosphorylation of E`CaCa with
ATP and release of ADP (reactions G and H), undergoes an isomeric
transition (reaction I) to a state (*E`-PCaCa) from which the
bound Ca can be released into the lumen of the
vesicles. *E`-PCaCa then undergoes hydrolytic cleavage
(reactions J to N) thereby completing a productive
cycle. Alternatively, E`-PCaCa can undergo hydrolysis before
releasing Ca
into the lumen of the vesicles, thereby
returning to the E`CaCa state (reactions F and E) from which the bound Ca
is released to
the medium outside the vesicles (reactions C to A) at
the end of an unproductive cycle.
Figure 9:
Diagram of a reaction scheme used for
computation of steady state velocities and intermediate states.
Bidirectional rate constants (s) of each sequential
step and the concentrations of substrates and products used in the
computations are given in the diagram. A detailed description of this
analysis and computation method is given in Inesi et al. (1988).
Computations based on this scheme
make it clear that a rise in lumenal Ca favors flux
through the alternate pathway which allows the intermediate to undergo
hydrolytic cleavage without releasing Ca
into the
lumen of the vesicles. Consequently, the overall coupling ratio is
reduced when lumenal Ca
rises ( Fig. 3and Table 1), as observed experimentally. An additional finding
predicted by the computations is a significant increase of the steady
state level of phosphorylated intermediate in the presence of high
lumenal Ca
( Table 1and Fig. 6). It is
also predicted by such computations (not shown) that, given the same
concentration of divalent cation in the lumen of the vesicles, the flux
through the branched pathway is affected by the affinity of the
phosphoenzyme for the divalent cation and by the kinetic constants of
each pathway. This explains the more favorable coupling ratios observed
in our experiments when Sr
is used instead of
Ca
, since the affinity of the phosphoenzyme for
Sr
is lower than the affinity for
Ca
. It also explains the more favorable ratios
observed when the lumen of the vesicles is maintained acid by replacing
sulfate with isothiocyanate (Fig. 7), since the affinity of the
phosphoenzyme for Ca
is lower at acid pH.
Our experiments raise a general question of whether it is reasonable to expect fixed coupling ratios in energy transducing enzymes, as predicted by schemes outlining rigid sequences of partial reactions. In principle, a protein equilibrated with various ligands will yield a statistical distribution of all possible species and isomers, depending on the appropriate binding and isomerization constants (Hill, 1991). In the case of an enzyme, each species should contribute to the overall reaction flux depending on its appropriate kinetic constant and its steady state concentration. In this paper we have considered only the simple case of one alternative pathway, assuming that all other possible branched reactions occur at negligible rates. This simple analysis is sufficient to explain the experimentally observed behavior and to underline a general point of interest regarding the coupling stoichiometry of energy transducing enzymes.
A final point of
interest is related to the physiology of cytosolic Ca removal by the transport ATPase associated with intracellular
organelles. Our present studies suggest that following a cytosolic
Ca
transient, such as that involved in contractile
activation of muscle fibers, the transport ATPase remains active until
enough Ca
is removed to reach a cytosolic
concentration below the level required for activation of the
Ca
ATPase. This mechanism would insure relaxation of
other Ca
-dependent cytosolic processes, such as
contraction of myofilaments. The calcium capacity of the organelles
would then be determined by the affinity of the enzyme lumenal sites
for Ca
and by the presence of
Ca
-binding proteins within the organelles.