Uncoupled ATPase Activity and Heat Production by the
Sarcoplasmic Reticulum Ca2+-ATPase
REGULATION BY ADP*
Leopoldo
de Meis
From the Instituto de Ciências Biomédicas, Departamento
de Bioquímica Médica, Universidade Federal do Rio de
Janeiro, Cidade Universitária, RJ 21941-590, Brazil
Received for publication, April 13, 2001, and in revised form, May 2, 2001
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ABSTRACT |
Sarcoplasmic reticulum vesicles of rabbit
skeletal muscle accumulate Ca2+ at the expense of ATP
hydrolysis. The heat released during the hydrolysis of each ATP
molecule varies depending on whether or not a Ca2+ gradient
is formed across the vesicle membrane. After Ca2+
accumulation, a part of the Ca2+-ATPase activity is not
coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995)
J. Biol. Chem. 270, 4361-4367). I now show that both
the heat produced during substrate hydrolysis and the uncoupled ATPase
activity vary depending on the ADP/ATP ratio in the medium. With a low
ratio, the Ca2+ transport is exothermic, and the formation
of the gradient increases the amount of heat produced during the
hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the
Ca2+ transport is endothermic, and formation of a gradient
increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the
synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis,
the Ca2+ efflux is exothermic (14-16 kcal/Ca2+
mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible
correlation of these findings with thermogenesis and anoxia is discussed.
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INTRODUCTION |
This work deals with two interconnected subjects: (i) the
mechanism of energy interconversion by enzymes and (ii) heat
generation, a process that plays a key role in the metabolic activity
and energy balance of the cell. The biological preparation used was vesicles derived from the sarcoplasmic reticulum of rabbit white skeletal muscle. These vesicles retain a membrane-bound
Ca2+-ATPase, which is able to interconvert different forms
of energy. During Ca2+ transport, the chemical energy
derived from ATP hydrolysis is used by the ATPase to pump
Ca2+ across the vesicle membrane, leading to the formation
of a transmembrane Ca2+ gradient (see reactions 1-6
forward in Figs. 1 and 2). In this process, chemical energy derived
from ATP hydrolysis is converted into osmotic energy. After
Ca2+ accumulation, the catalytic cycle of the enzyme can be
reversed, and the accumulated Ca2+ leaves the vesicles
through the Ca2+-ATPase synthesizing ATP from ADP and
Pi (read reactions 6 to 1 backward in Figs. 1 and 2).
During synthesis, osmotic energy is converted back into chemical energy
(1-6). In the steady state, the Ca2+ concentrations inside
the vesicles and in the assay medium remain constant, but the ATPase
operates simultaneously forward (ATP hydrolysis and Ca2+
uptake) and backwards (Ca2+ efflux and ATP synthesis), and
chemical and osmotic energy are continuously interconverted by the ATPase.
The catalytic cycle of the ATPase varies depending on the
Ca2+ concentration in the vesicle lumen. When the free
Ca2+ concentration inside the vesicles is kept in the
micromolar range, the reaction cycle flows as shown in Fig. 1 (2-5).
The main feature of this cycle is that the hydrolysis of each ATP
molecule is coupled with the translocation of two Ca2+ ions
across the membrane (4-7). This was best measured in pre-steady state
experiments in which the luminal Ca2+ has yet to rise
(8-10). The enzyme cycles through two more sets of intermediary
reaction when intact vesicles are used, and the Ca2+
concentration inside the vesicles rises to the millimolar range (see
Fig. 2). These are ramifications of the catalytic cycle and are denoted
as dashed lines in Fig. 2. In one of them, a part of the Ca2+ accumulated by the vesicles leaks through the
enzyme without catalyzing the synthesis of ATP. This is referred to as
uncoupled Ca2+ efflux and is represented by reactions 7-9
in Fig. 2 (11-14). In 1995, Yu and Inesi (10) and later Fortea
et al. (15) observed that the progressive rise in the
luminal Ca2+ concentrations promotes another ramification
of the catalytic cycle sequence leading to ATP hydrolysis without
Ca2+ translocation. According to these authors, the
uncoupled ATP hydrolysis is derived from the cleavage of the
phosphoenzyme form 2Ca:E1~P (reaction
10 in Fig. 2).
In recent reports (16-20), it was shown that chemical and osmotic
energy are not the only two forms of energy interconverted by the
ATPase. During the steady state, a fraction of both chemical and
osmotic energy is converted by the ATPase into heat. The total amount
of energy released during ATP hydrolysis is always the same, but the
fraction of the total energy that is converted into either chemical or
osmotic energy or heat seems to be modulated by the ATPase. The main
experimental finding that led to this conclusion was that the amount of
heat released during the hydrolysis of each ATP molecule varies
depending on whether or not a transmembrane gradient is formed across
the vesicle membrane. In the absence of a Ca2+ gradient
(leaky vesicles; see Fig. 1) between 10 and 12 kcal are released for
each mol of ATP cleaved, and in the presence of a Ca2+
gradient (intact vesicles; see Fig. 2) the amount of heat released increases to the range of 20-24 kcal/mol of ATP cleaved. At present, it is not clear why the amount of heat produced during the hydrolysis of each ATP molecule increases after Ca2+ accumulation. One
of the catalytic routes involved in heat production seems to be the
uncoupled Ca2+ efflux (20). In this case, the energy
derived from ATP hydrolysis is first converted into osmotic energy
(reactions 1-4 in Fig. 2), and then during the uncoupled
Ca2+ efflux (reactions 7-9), osmotic energy is converted
into heat. This work now raises the possibility that the uncoupled ATP
hydrolysis discovered by Yu and Inesi (10) may represent a second route of heat production. If the hydrolysis of ATP is completed before Ca2+ translocation through the membrane (reaction 10 in
Fig. 2), then there is no conversion of chemical into osmotic energy,
and during catalysis more chemical energy should be left available to
be converted into heat. In order to test this hypothesis, I measured the rates of uncoupled Ca2+ efflux and uncoupled ATP
hydrolysis in the presence of different ADP concentrations. It is known
(3, 4, 21) that reaction 2 in Fig. 2 is highly reversible
(Keq
1). Therefore, during catalysis, the
fraction of enzyme that accumulates in the form 2Ca:E1~P depends on the ratio between the ADP
and ATP concentrations available in the medium. While ATP
phosphorylates the enzyme form 2Ca:E1 (reaction
2 forward), ADP drives the reversal of the reaction converting
2Ca:E1~P back to
2Ca:E1. The rise in the intravesicular Ca2+ concentration promotes inhibition of the ATPase
activity and an increase in the steady state level of the enzyme form
2Ca:E1~P. This is referred to in the
bibliography as back inhibition (1, 3-6), and it is the increase of
2Ca:E1~P level noted during the back
inhibition that promotes the uncoupled ATP hydrolysis through reactions
2 and 10 in Fig. 2 (10, 15). Because reaction 2 is highly reversible,
it should be expected that an increase of the ADP concentration in the
medium should prevent the accumulation of
2Ca:E1~P, and if in fact the uncoupled ATP
hydrolysis proceeds through reaction 10 (10, 15) and if this cleavage
produces more heat than the coupled ATP hydrolysis (reactions 3-5 in
Figs. 1 and 2) as I hypothesize, then both the uncoupled ATP hydrolysis and the amount of heat produced during the cleavage of each ATP molecule should decrease in the presence of a high ADP concentrations. This working hypothesis was tested in this report using different ATP-regenerating systems.
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MATERIALS AND METHODS |
Sarcoplasmic Reticulum Vesicles--
These were derived from the
longitudinal sarcoplasmic reticulum of rabbit hind leg white skeletal
muscle and were prepared as previously described (22). The vesicles
were stored in liquid nitrogen until use. The efflux of
Ca2+ measured with these vesicles was not altered by
ryanodine, indicating that they did not contain significant
amounts of ryanodine-sensitive Ca2+ channels. The vesicles
also did not exhibit the phenomenon of Ca2+-induced
Ca2+ release found in the heavy fraction of the
sarcoplasmic reticulum (13, 22).
Ca2+-loaded Vesicles--
Vesicles were preloaded
with either 40Ca2+ or
45Ca2+ using different assay media as described
in the figure legends for Figs. 3 to 10. After 30-40 min of
incubation at 35 °C, the vesicles were centrifuged at 40,000 × g for 40 min, the supernatant was discarded, and the pellet
was kept in ice and resuspended before starting the experiment in a
small volume of the loading mixture to reach the final vesicle concentration of 1.0-1.5 mg of protein/ml. The vesicles loaded with
45Ca2+ were used for measurement of
Ca2+ efflux, and the vesicles loaded with
40Ca2+ were used for calorimetric measurements
and for measurement of ATP synthesis from ADP and
32Pi.
Ca2+ Uptake, Ca2+ Efflux, and
Ca2+in
Ca2+out
Exchange--
This was measured by the filtration method (23). For
45Ca uptake, trace amounts of 45Ca were
included in the assay medium. For 45Ca efflux, vesicles
previously loaded with 45Ca were used. The reaction was
arrested by filtering samples of the assay medium in Millipore filters.
After filtration, the filters were washed five times with 5 ml of 3 mM La(NO3)3, and the radioactivity remaining on the filters was counted using a liquid scintillation counter. For the Ca2+in
Ca2+out exchange the assay medium was divided
in two samples. Trace amounts of 45Ca2+ were
added to only one of the samples, and the reaction was started by the
simultaneous addition of vesicles to the two media. The sample
containing the radioactive Ca2+ was used to determine the
incubation time where the vesicles were filled and the steady state of
45Ca2+ uptake was reached. The rate of
Ca2+in
Ca2+out
exchange was measured after that steady state was reached by adding a
trace amount of 45Ca2+ to the second sample
containing vesicles loaded with nonradioactive Ca2+. The
exchange of the radioactive Ca2+ from the medium with the
nonradioactive Ca2+ contained inside the vesicles was
measured by filtering samples of the assay medium in Millipore filters
10, 20, 30, 40, 60, and 120 s after the addition of
45Ca2+.
ATPase Activity and Cleavage of
PEP,1 Glc-6-P, and
Fru-1,6-P--
These were assayed using either a colorimetric
method or by measuring the release of 32Pi from
either [
-32P]ATP, [32P]Glc-6-P, or
[1-32P]Fru-1,6-P (24-26). The
32Pi produced was extracted from the medium
with ammonium molybdate and a mixture of isobutyl alcohol and benzene.
When the colorimetric method was used, Pi was not included
in the assay medium. In the various experimental conditions used, the
same results were obtained with either the colorimetric method or with
the use of radioactive substrate, regardless of the ATP concentrations
and ATP-regenerating system used. The values of ATPase activity shown
in the figures and tables are the
Ca2+-dependent activity responsible for
Ca2+ transport. The Mg2+-dependent
activity was measured in the presence of 2 mM EGTA. The
Ca2+-dependent activity was determined by
subtracting the Mg2+-dependent activity from
the activity measured in the presence of both Mg2+ and
Ca2+. In the different experimental conditions used, the
Mg2+-dependent activity represented 2-10% of
the total activity measured.
ATP Synthesis--
This was measured using
32Pi as previously described (24).
Heat of Reaction--
This were measured using an OMEGA
Isothermal Titration Calorimeter from Microcal Inc. (Northampton, MA)
(16-20). The calorimeter cell (1.5 ml) was filled with reaction
medium, and the reference cell was filled with Milli-Q water. After
equilibration at 35 °C, the reaction was started by injecting
vesicles into the reaction cell, and the heat change during either
Ca2+ uptake or Ca2+ efflux was recorded for
20-30 min. The volume of vesicle suspension injected in the cell
varied between 0.02 and 0.03 ml. The heat change measured during the
initial 2 min after vesicle injection was discarded in order to avoid
artifacts such as the heat derived from the dilution of the medium
containing the loaded vesicles into the efflux medium and the binding
of ions to the Ca2+-ATPase. The duration of these events is
less than 1 min. The calorimetric enthalpy
(
Hcal) was calculated by dividing the amount
of heat released by the amount of either substrate hydrolyzed or
Ca2+ released by the vesicles. The units used were moles
for substrate hydrolyzed and Ca2+ released and kcal for the
heat released. A negative value indicates that the reaction is
exothermic, and a positive value indicates that it is endothermic.
Procedures--
All experiments were performed at
35 °C. In a typical experiment, the assay medium was divided into
five samples, which were used for the simultaneous measurement of
Ca2+ uptake, Ca2+in
Ca2+out exchange, substrate hydrolysis, ATP
synthesis, and heat release. The syringe of the calorimeter was filled
with the vesicles, and the temperature difference between the syringe and the reaction cell of the calorimeter was allowed to equilibrate, a
process that usually took between 8 and 12 min. After equilibration, the reaction was started by injecting the vesicles into the reaction cell. During equilibration, the vesicles used for measurements of
Ca2+ uptake, Ca2+in
Ca2+out exchange, ATP hydrolysis, and ATP
synthesis were kept at the same temperature, length of time, and
protein dilution as the vesicles kept in the calorimeter syringe. The different reactions were started simultaneously using either empty vesicles or Ca2+-loaded vesicles. For the experiments where
the unidirectional Ca2+ efflux was measured, the heat
released during the efflux was corrected for the heat derived from both
the binding of Ca2+ to EGTA and the heat derived from the
formation of Glc-6-P from ATP and glucose as previously described (20).
NaN3, an inhibitor of ATP synthase, and
P1,P5-di(adenosine-5') pentaphosphate, a
specific inhibitor of adenylate kinase, were added to the assay medium
in order to avoid interference from possible contamination of the
sarcoplasmic reticulum vesicles with these enzymes.
The free Ca2+ concentration in the medium was calculated
using the association constants of Schwartzenbach et al.
(27) in a computer program described by Fabiato and Fabiato (28) and modified by Sorenson et al. (29).
ATP-regenerating System--
A large excess of pyruvate kinase,
hexokinase, or phosphofructokinase was used in order to assure that ATP
was regenerated at a faster rate than it was cleaved by the
Ca2+-ATPase. In control experiments, the rates of substrate
hydrolysis and Ca2+ uptake were measured in the presence of
different concentrations of the ATP-regenerating enzymes, and the
concentration of enzyme used in all experiments described was 5-10
times higher than that needed for maximal activity (25, 26).
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RESULTS |
Ca2+ Transport in the Presence of 1 mM ATP
and PEP
Most of the measurements performed in this work were made after
the Ca2+ uptake reached the steady state. When the vesicles
are still being filled, the rate of Ca2+ uptake measured
represents a balance between the Ca2+ pumped inside the
vesicles by the ATPase and the rate of Ca2+ that leaves the
vesicles driven by the gradient formed across the membrane. During the
initial minutes of incubation, these two rates are different and cannot
be measured separately. Thus, the stoichiometry between the fluxes of
Ca2+ through the membrane and the rates of either ATP
cleavage or ATP synthesis cannot be evaluated with precision. After the
steady state is reached, the rate of efflux is the same as that of
Ca2+ uptake, and by measuring the rate of
Ca2+in
Ca2+out
exchange it is possible to determine the value of the two rates. The
exchange represents the fraction of Ca2+ that leaves the
vesicles and is pumped back inside the vesicles by the ATPase.
The initial velocities of Ca2+ uptake measured with 1 mM ATP and 50 µM ATP plus PEP as the
ATP-regenerating system were the same (see Fig. 3A).
However, when the steady state was reached, the amount of
Ca2+ retained by the vesicles with PEP was larger than that
accumulated with 1 mM ATP. In six experiments, the steady
state levels of Ca2+ uptake were 3.90 ± 0.25 µmol/mg with PEP and 2.73 ± 0.07 µmol/mg with 1 mM ATP. These values are the mean ± S.E. The rate of
Ca2+in
Ca2+out
exchange measured in the presence of PEP was slower than that measured
with 1 mM ATP and no ATP-regenerating system (Table
I). The time course of ATP hydrolysis was
found to vary depending on the condition used. With the use of PEP, the
rate of hydrolysis did not vary with the incubation interval, being
practically the same before and after the steady state of Ca2+ uptake was reached. With 1 mM ATP,
however, a significant decrease in the ATPase activity was detected
after the vesicles were filled with Ca2+ (see Fig.
3B). This difference was probably related to the
accumulation of ADP in the medium during the course of the reaction
that drives reaction 2 in Fig. 2 backwards (1-6, 13, 21). The apparent Km of the enzyme form 2Ca:E1
for ATP is in the range of 1-3 µM, and
Km of the form 2Ca:E1~P for
ADP is in the range of 10-30 µM (2, 3). In the
incubation interval of 10-40 min (see Fig. 3), 15-40% of the ATP
added was cleaved (i.e. the ADP concentration rose from 0.15 to 0.40 mM), being therefore sufficient to promote
the reversal of reaction 2 shown in Fig. 2. In the presence of PEP,
there is practically no accumulation of ADP in the medium.
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Table I
Rates of ATP hydrolysis, ATP synthesis, and Ca2+in Ca2+out exchange at steady state
The assay medium composition and experimental conditions were as in
Figs. 3 and 4. The Ca2+/ATP ratio was calculated by dividing
the rate of Ca2+in Ca2+out exchange
by the rate of net hydrolysis of ATP. The values in the table are
means ± S.E. of six experiments.
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The Ca2+ concentration in the lumen of intact vesicles
reaches the millimolar range in a few seconds after the transport is initiated (1-6, 8, 9). This triggers the reversal of the catalytic
cycle of the ATPase (30-32), during which Ca2+ leaves the
vesicles through the ATPase, and ATP is synthesized from ADP and
Pi (see Fig. 3B). With the use of PEP, there was no measurable synthesis of ATP due to the absence of ADP, one of the
substrates needed for the synthesis. With 1 mM ATP, the amount of ADP accumulated during steady state was sufficient to maximally activate the synthesis of ATP from ADP and Pi
(30, 31), and about 10% of the ATP cleaved during steady state was synthesized back due to the reversal of the Ca2+ pump
(Table I).
Knowing the rates of Ca2+in
Ca2+out, ATP hydrolysis and ATP synthesis it
was possible to estimate the steady state values of the following.
The Net ATP Hydrolysis--
This represents the true amount of ATP
cleaved to maintain the Ca2+ gradient formed across the
vesicle membrane. This was calculated by subtracting the rate of ATP
synthesis from the rate of ATP hydrolysis (Table I).
The Ratio between the Rates of ATP Hydrolysis and ATP
Synthesis--
This ratio gives a measure of the degree of energy
conservation of the system (3, 30-32). The more ATP is synthesized,
the smaller is the ratio between the rates of hydrolysis and synthesis and the more energy is conserved by the system; i.e. the
steady state can be conserved for a longer period of time because the net decline of the ATP concentration in the medium proceeds at a slower
rate. With the use of PEP as an ATP-regenerating system, there is no
energy conservation, because none of the substrate cleaved is
synthesized back by the system. With 1 mM ATP, in six experiments the ratio between the rates of ATP hydrolysis and ATP
synthesis was 11 ± 2.
The Ratio between the Rates of Ca2+ Uptake and ATP
Hydrolysis--
This was calculated by dividing the rate of
Ca2+in
Ca2+out
exchange by the rate of net ATP hydrolysis (Table I). Both with ATP and
PEP, the values of the Ca2+/ATP ratio were smaller than 2, but with 1 mM ATP the coupling ratio was higher than that
measured with PEP, suggesting that the presence of ADP in the medium
promotes a better coupling of the system.
The Rates of Ca2+ Efflux Coupled with the Synthesis of
ATP (Reactions 5 to 2 Backwards in Fig. 2)--
In different
laboratories, it has already been shown that the release of two
Ca2+ ions from the vesicles drives the synthesis of one ATP
molecule (1-6). The coupled Ca2+ efflux was therefore
calculated by multiplying the rate of ATP synthesis by 2, and the
difference between the rate of Ca2+in
Ca2+out exchange and the coupled
Ca2+ efflux represents the uncoupled Ca2+
efflux (reactions 7-9 in Fig. 2). With the use of PEP there was no ATP
synthesis. Therefore, all of the efflux measured was uncoupled. With 1 mM ATP, about one-third of the Ca2+ that leaves
the vesicle during the steady state is coupled with the synthesis of
ATP (Table II).
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Table II
Rates of coupled and uncoupled ATPase activity and Ca2+ efflux
The assay medium composition and experimental conditions were as in
Figs. 3 and 4 and Table I. The values in the table are means ± S.E. of six experiments.
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The Rates of ATP Hydrolysis Coupled and Uncoupled with the
Translocation of Ca2+--
For these calculations, I used
the values of net ATP hydrolysis and the stoichiometry of two
Ca2+ ions pumped for each ATP molecule cleaved. Thus, the
rate of Ca2+in
Ca2+out exchange shown in Table I divided by 2 gives the rate of coupled ATP hydrolysis, i.e. the ATP
cleaved to pump back the Ca2+ that leaves the vesicles
during the Ca2+in
Ca2+out exchange (reactions 1-5 in Fig. 2).
The difference between the total net ATP hydrolysis and the coupled ATP
hydrolysis gives the value of the uncoupled ATPase activity (reactions
2 and 10 in Fig. 2). With the use of PEP, about 90% of the ATP cleaved
was not coupled with the transport of Ca2+, while with 1 mM ATP about 69% of the net ATPase activity was uncoupled
(Table II). These data confirm the findings of Yu and Inesi (10) and
Fortea et al. (15) that after Ca2+ accumulation,
a significant fraction of the ATP cleaved by the Ca2+-ATPase is not coupled with the translocation of
Ca2+ through the membrane and in addition shows that the
rate of the uncoupled ATPase activity decreases when ADP accumulates in
the medium.
Ca2+ Transport in the Presence of High ADP
Concentration
In order to measure the Ca2+ transport in the presence
of a high ADP/ATP ratio, I used sugar phosphates as the
ATP-regenerating system. In previous reports (25, 26), it was shown
that the Ca2+-ATPase can use both Glc-6-P and hexokinase or
Fru-1,6-P and phosphofructokinase as ATP-regenerating systems. The
affinity of the Ca2+-ATPase for ATP is sufficiently high
(Ka = 10
6 M)
to permit the formation of the enzyme-substrate complex even in the
presence of the very low concentration of ATP formed from ADP and
either Glc-6-P or Fru-1,6-P. During Ca2+ uptake, the ADP
formed from ATP is phosphorylated by the sugar phosphate in order to
maintain the equilibrium concentration of ATP. Thus, in steady state
conditions, the Ca2+ transport proceeds as if it was
supported by the cleavage of the sugar phosphate as shown for Glc-6-P
by the addition of Reactions 1 and 2.
The difference between the use of sugar phosphate or PEP as
ATP-regenerating systems is the amount of ADP available in the medium
during the reaction. While with PEP there is practically no ADP
available, with the sugar phosphate most of the nucleotide in the
medium is in the form of ADP (Table III).
The initial rates of Ca2+ transport and steady state level
of Ca2+ accumulation obtained with the use of either
Glc-6-P or Fru-1,6-P were 2-4-fold smaller than those measured with
either 1 mM ATP or 10 µM ATP and PEP (compare
Figs. 3A and 4A). Comparing the two sugar
phosphates, the vesicles were able to accumulate more Ca2+
and at a faster rate with Fru-1,6-P (see Fig. 4A), and when
the steady state was reached, the amount of Ca2+ retained
by the vesicles in six experiments with Fru-1,6-P and Glc-6-P was
1.37 ± 0.03 and 1.00 ± 0.03 µmol/mg, respectively. The
rates of Ca2+in
Ca2+out exchange measured at steady state with
Glc-6-P and Fru-1,6-P were practically the same as those measured with 1 mM ATP (see Fig. 4A and Table I), but the
rates of Ca2+-dependent ATP hydrolysis were
slower than those observed with 1 mM ATP (Table I). In the
presence of sugar phosphate, a significant amount of ATP was
synthesized during the steady state. The reactions involved in the
regeneration of ATP shown above flow both forward and backwards, and
the radioactive phosphate of the [
-32P]ATP synthesized
during reversal of the Ca2+ pump is continuously
transferred to either glucose or Fru-6-P added to the assay
medium, forming [32P]Glc-6-P and
[32P]Fru-1,6-P, which are diluted in the large pool of
nonradioactive sugar phosphate available in the medium. In contrast to
the rates of hydrolysis, the rates of ATP synthesis measured with the
sugar phosphate were similar to those measured with 1 mM
ATP (Table I). As a result, the ratios between the rates of substrate
hydrolysis and synthesis were smaller than those measured with ATP.
This new finding shows that a high ADP concentration favors the energy conservation of the Ca2+ transport system. In fact, while
with 1 mM ATP the ratio between the rates of ATP cleavage
and ATP synthesis was 11.0, with the use of Glc-6-P and Fru-1,6-P in
six experiments the values found were 5.6 ± 0.7 and 2.4 ± 0.2, respectively. When the rates of Ca2+ efflux and ATP
synthesis were compared, it was found that the rates of coupled and
uncoupled Ca2+ efflux measured with 1 mM ATP
and Glc-6-P were practically the same, but with Fru-1,6-P there was a
significant predominance of the coupled efflux over the uncoupled
Ca2+ efflux (Table II). However, the most interesting
finding was that the uncoupled ATPase activity was practically
abolished in the presence of both Glc-6-P and Fru-1,6-P (Table II), and
as a result, the ratio between net ATP cleavage and
Ca2+in
Ca2+out
exchange was 2 (Table I). These findings show that the ramification of
the catalytic cycle proposed by Yu and Inesi (10) is abolished when the
ADP concentration is higher than that of ATP, thus providing an
experimental condition that permits verifying whether or not the amount
of heat produced during the hydrolysis of the enzyme form
2Ca:E1~P (reaction 10 in Fig. 2) is larger
than that measured during the hydrolysis of E2-P
(reactions 5 and 6 in Figs. 1 and
2).
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Table III
ATP concentration in media containing different concentrations of ADP
and sugar phosphate
The ATP concentrations shown in the table were calculated as previously
described (25, 26) using a Keq of 6.27 × 10 4 for the reaction Glc-6-P + ADP ATP + glucose and a Keq of 10 3 for the reaction
Fru-1,6-P + ADP ATP + Fru-6-P. Notice that in all
experiments performed, a small concentration (50 µM) of
either glucose or Fru-6-P was included in the medium. This was done to
facilitate the calculations of ATP in the medium and to avoid the
situation where the reaction would flow in only one direction due to
the lack of one of the reactants.
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Fig. 1.
The catalytic cycle of the
Ca2+-ATPase in the absence of a Ca2+
concentration gradient. The sequence includes two distinct enzyme
conformations, E1 and E2.
The Ca2+ binding sites in the E1
form face the external surface of the vesicle and have a high affinity
for Ca2+ (Ka = 10 6
M at pH 7). In the E2 form, the
Ca2+ binding sites face the vesicle lumen and have a low
affinity for Ca2+ (Ka = 10 3 M at pH 7). The enzyme form
E1 is phosphorylated by ATP but not by
Pi, and, conversely, the enzyme form
E2 is phosphorylated by Pi but not
by ATP. When the Ca2+ concentration on the two sites of the
membrane is inferior to 50 µM, reaction 4 is
irreversible, and this forces the sequence to flow forward from
reaction 1 to 6. For details, see Refs. 2-4.
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Fig. 2.
The catalytic cycle of the
Ca2+-ATPase in the presence of a transmembrane
Ca2+ concentration gradient. The characteristics of
the enzyme forms E1 and
E2 are the same as those of Fig. 1. This
sequence is observed when the Ca2+ concentration on the
outer surface of the membrane is inferior to 50 µM and
the concentration in the vesicle lumen is higher than 1 mM.
The high intravesicular Ca2+ concentration permits the
reversing of reactions 4 and 3. As a result, the catalytic cycle can be
reversed, leading to ATP synthesis and Ca2+ efflux
(reactions 5 to 1 backwards). The uncoupled Ca2+ efflux is
mediated by reactions 7-9 flowing forward. The uncoupled ATPase
activity is mediated by reaction 10 (10-15).
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Control Experiments
The following experiments demonstrate that the Ca2+
accumulation, Pi production, and ATP synthesis measured in
Fig. 4 and Tables I and II were in fact derived from the hydrolysis of
ATP generated by ADP and either Glc-6-P or Fru-1,6-P: (i) there was no
Ca2+ uptake nor Pi production if either the
sugar phosphate (Glc-6-P or Fru-1,6-P) or the enzymes needed for
regenerating ATP (hexokinase or phosphofructokinase) were omitted from
the medium; (ii) there was no Ca2+ uptake nor
Pi production when the concentrations of glucose and Fru-6-P in the medium were raised from 50 µM to 5 mM (this promotes a decrease of the calculated ATP
concentration in the medium from 3.1 to 0.03 µM in the
case of Glc-6-P and from 5.0 to 0.05 µM in the case of
Fru-1,6-P (25, 26)); (iii) thapsigargin, a highly specific inhibitor of
the sarcoplasmic reticulum Ca2+-ATPase, inhibited the
Ca2+ transport supported by 1 mM ATP and by the
three different ATP-regenerating systems used in Figs.
3 and 4;
and (iv) there was no ATP synthesis if the vesicles were rendered leaky
by the addition of the Ca2+-ionophore A23187 regardless of
whether 1 mM ATP or a sugar phosphate was used as an
ATP-regenerating system.

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Fig. 3.
Ca2+ uptake,
Ca2+in Ca2+out
exchange (A), ATP hydrolysis, and ATP synthesis
(B) in the presence and absence of PEP. The assay
medium composition was 50 mM MOPS-Tris buffer (pH 7.0), 2 mM MgCl2, 120 µM
CaCl2, 100 µM EGTA, 10 mM
Pi, 100 mM KCl, 5 mM
NaN3, 10 µM
P1,P5-di(adenosine-5') pentaphosphate and
either 1 mM ATP ( , ) or 50 µM ATP, 2 mM PEP, and 10 units/ml pyruvate kinase ( , ). The
reaction was performed at 35 °C and was started by the addition of
vesicles, 20 µg protein/ml. A, Ca2+ uptake
(open symbols and left
ordinate) and Ca2+in Ca2+out exchange (closed
symbols and right ordinate);
B, ATPase activity (open symbols and
left ordinate) and ATP synthesis
(closed symbols and right
ordinate). For the media containing 1 mM ATP and
10 µM ATP, the calculated free Ca2+
concentrations in the media were 10.1 and 15.7 µM,
respectively, before the addition of vesicles and decreased to an
average value of 0.4 and 0.2 µM, respectively, after the
vesicles were filled with Ca2+ (steady state). Values are
means ± S.E. of six experiments.
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Fig. 4.
Ca2+ uptake,
Ca2+in Ca2+out
exchange (A), ATP hydrolysis, and ATP synthesis
(B) using Fru-1,6-P and Glc-6-P as an ATP-regenerating
system. The assay medium composition was 50 mM
MOPS-Tris buffer (pH 7.0), 2 mM MgCl2, 120 µM CaCl2, 100 µM EGTA, 10 mM Pi, 100 mM KCl, 50 µM ADP, 5 mM NaN3, 10 µM P1,P5-di(adenosine-5')
pentaphosphate, and either 5 mM Glc-6-P, 50 µM glucose and 10 units/ml hexokinase ( , ), or 5 mM Fru-1,6-P, 50 µM Fru-6-P, and 10 units/ml
phosphofructokinase ( , ). The reaction was performed at 35 °C
and was started by the addition of vesicles, 20 of µg protein/ml.
A, Ca2+ uptake (open
symbols and left ordinate) and
Ca2+in Ca2+out
exchange (closed symbols and right
ordinate); B, ATPase activity (open
symbols and left ordinate) and ATP
synthesis (closed symbols and right
ordinate). Both with Glc-6-P and Fru-1,6-P, the calculated
free Ca2+ concentration in the medium was 15.7 µM before the addition of vesicles and decreased to 5.7 µM in the presence of Glc-6-P and to 4.0 µM
in the presence of Fru-1,6-P after the vesicles were filled with
Ca2+ (steady state). Values are means ± S.E. of six
experiments.
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Back Inhibition and Heat Production in the Presence and Absence of
a Ca2+ Gradient
In these experiments, the rate of heat release and the rate of
substrate hydrolysis were measured simultaneously in leaky vesicles (no
gradient) and intact vesicles (gradient). For intact vesicles, the
values of hydrolysis were corrected for the ATP or sugar phosphate
synthesized back at the different incubation intervals (net
hydrolysis). In agreement with previous reports (1-6, 30-32), there
was no synthesis of ATP in the absence of a Ca2+ gradient.
The rate of hydrolysis of the leaky vesicles was always higher than
that measured with intact vesicles (gradient). This difference is
related to the back inhibition promoted by the rise of the
intravesicular Ca2+ concentration (1, 3-6, 13). I now show
that the back inhibition is modulated by ADP (Table
IV). With the use of PEP as the
ATP-regenerating system (no ADP available) the rise in the
intravesicular Ca2+ concentration promoted a 18.8%
inhibition of ATPase activity. In the presence of ADP and depending on
the substrate used, the back inhibition increased to the range of
54.8-75.7%. This indicates that in the absence of ADP, the decrease
of the coupled ATPase activity (reactions 1-5 in Fig. 2) is
compensated by the fast rate of uncoupled ATPase activity (reaction
10). As the ADP concentration in the medium rises, the uncoupled ATPase
activity is arrested, and the back inhibition of the coupled ATPase
becomes more visible.
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Table IV
Back inhibition, release, and absorption of heat during Ca2+
transport: Hcal values
Other additions to the assay medium and experimental conditions were as
in Figs. 5 and 6. Values are means ± S.E. of the number of
experiments (n) shown in the table.
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The transport of Ca2+ was exothermic when measured in the
presence of either 1 mM ATP or 10 µM ATP and
2 mM PEP (Table IV and Fig.
5). The amount of heat released in the
presence and absence of a Ca2+ gradient was proportional to
the amount of ATP hydrolyzed, both during the initial incubation
intervals, where the vesicles accumulate Ca2+, and after
prolonged incubation intervals, where the vesicles were filled and the
steady state was reached. This could be visualized by plotting the heat
release as a function of the amount of ATP hydrolyzed (Fig.
5C). In earlier reports (16-20), it was found that the heat
released for each ATP molecule hydrolyzed by intact vesicles was larger
than that measured with leaky vesicles. This was confirmed in Table IV.
I now show that the difference between intact and leaky vesicles is
also observed when the ATP cleaved is continuously regenerated by PEP.
The amount of heat produced during the hydrolysis of each PEP molecule
cleaved in the presence of a gradient was 2 times larger than that
measured in the absence of a gradient (Fig. 5 and
Hcal values in Table IV). A different result
was obtained when Fru-1,6-P and Glc-6-P were used as the
ATP-regenerating system. The Ca2+ transport supported by
the sugar phosphate was endothermic, and the amount of heat absorbed
from the environment with intact vesicles was larger than that measured
with leaky vesicles (Fig. 6 and Table
IV). In a control experiment, the hydrolysis of Glc-6-P and Fru-1,6-P
catalyzed by leaky vesicles and hexokinase was compared with the
hydrolysis catalyzed by alkaline phosphatase and no hexokinase (Fig.
7). In the first condition, the sugar
phosphate had to transfer its phosphate first to ADP, forming ATP, and
then the ATP formed was cleaved by the ATPase. With the use of alkaline
phosphatase, the sugar phosphate was directly cleaved by the enzyme. In
seven experiments, the
Hcal values obtained
for the cleavage of Fru-1,6-P and Glc-6-P by alkaline phosphatase were
+0.78 ± 0.14 and +0.57 ± 0.04, respectively. These values
are the same as those measured with leaky vesicles in Table IV,
indicating that the difference in heat absorption measured in the
presence and absence of a Ca2+ gradient in Table IV and
Fig. 6 was related to Ca2+ accumulation by the vesicles and
not to the transfer of phosphate from the sugar phosphate to ADP.

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Fig. 5.
Heat released during ATP hydrolysis using PEP
as the ATP-regenerating system in the presence ( ) and absence ( )
of a Ca2+ gradient. The assay medium composition was
50 mM MOPS-Tris buffer (pH 7.0), 2 mM
MgCl2, 100 µM CaCl2, 100 µM EGTA, 10 mM Pi, 100 mM KCl, 10 µM ATP, 2 mM PEP, 10 units/ml phosphofructokinase, 5 mM NaN3, 10 µM P1,P5-di(adenosine-5')
pentaphosphate without ionophore ( ) or with 10 µM
ionophore A23187 ( ). The reaction was performed at 35 °C and was
started by the addition of vesicles, 10 µg of protein/ml.
A, ATPase activity; B, heat measurement. In
C, data of A and B were replotted. In
the absence of ionophore, the vesicles accumulated 3.55 ± 0.27 µmol of Ca2+/mg of protein. Note that both the ATPase
activity and heat release were measured 2 min after starting the
reaction with the vesicles (see "Materials and Methods"). There was
no Ca2+ uptake in presence of the ionophore. The calculated
free Ca2+ concentration in the media was 4.03 µM before the addition of vesicles, and in intact
vesicles the concentration decreased to 0.42 µM after the
vesicles were filled with Ca2+. Values are means ± S.E. of five experiments.
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Fig. 6.
Heat absorption during ATP hydrolysis using
Fru-1,6-P as the ATP-regenerating system in the presence ( ) and
absence ( ) of a Ca2+ gradient. The assay medium
composition was 50 mM MOPS-Tris buffer (pH 7.0), 2 mM MgCl2, 100 µM
CaCl2, 100 µM ADP, 20 mM
Pi, 100 mM KCl, 5 mM Fru-1,6-P, 50 µM Fru-6-P, 10 units/ml pyruvate kinase, 5 mM
NaN3, 10 µM
P1,P5-di(adenosine-5') pentaphosphate without
ionophore A23187 ( ) or with 10 µM ionophore A23187
( ). The reaction was performed at 35 °C and was started by the
addition of vesicles, 50 µg protein/ml. A, ATPase
activity; B, heat measurement. In C, data of
A and B were replotted. In the absence of
ionophore, the vesicles accumulated 1.41 ± 0.03 µmol of
Ca2+/mg of protein. There was no Ca2+ uptake in
the presence of the ionophore. The free Ca2+ concentration
in the medium was 60.6 µM before the addition of
vesicles, and in intact vesicles concentration decreased to the range
of 17.9 µM after steady state. Values are means ± S.E. of three experiments.
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Fig. 7.
Heat released during Glc-6-P hydrolysis
catalyzed by either leaky vesicles ( ) or alkaline phosphatase
( ). For leaky vesicles, the assay medium composition was 50 mM MOPS-Tris buffer (pH 7.0), 2 mM
MgCl2, 100 µM CaCl2, 100 µM ADP, 20 mM Pi, 100 mM KCl, 5 mM Glc-6-P, 10 units/ml hexokinase, 5 mM NaN3, 10 µM
P1,P5-di(adenosine-5') pentaphosphate, and 10 µM ionophore A23187 ( ). For the alkaline phosphatase,
the assay medium composition was 50 mM MOPS-Tris buffer (pH
7.0), 1 mM MgCl2, 5 mM Glc-6-P and
1 unit/mg alkaline phosphatase. Shown is a representative experiment
performed at 35 °C.
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Reversal of the Ca2+ Pump and Energy Interconversion
during Unidirectional Ca2+ Efflux
In an early report, Rossi et al. (33) observed that the
stoichiometry of Ca2+ transport may vary depending on the
substrate used to energize the Ca2+ pump. These authors
found a coupling ratio of 1 with the use of either
p-nitrophenylphosphate, methylumbelliferylphosphate, or
furylacryloylphosphate. The aim of the following experiments was to
verify whether or not the molar ratio between Ca2+ efflux
and ATP synthesis measured in the presence of sugar phosphates was in
fact 2, as assumed for the calculations of the coupled and uncoupled
activities in Table II. The experiment of Fig.
8 was performed using Fru-1,6-P as the
ATP-regenerating system. After the vesicles were loaded with
Ca2+, an aliquot of the assay medium was mixed with an
excess of EGTA and an excess of Fru-6-P in order to drastically
decrease the concentrations of both Ca2+ and ATP in the
medium. Following this maneuver, the pumping was arrested, and the
Ca2+ accumulated by the vesicles leaked to the
medium at a fast rate. The Ca2+ efflux was coupled with the
synthesis of ATP, and both the rate of Ca2+ efflux and ATP
synthesis were impaired when thapsigargin was included in the efflux
media. In eight different experiments, using either Glc-6-P or
Fru-1,6-P as the ATP-regenerating system, the ratio between the rates
of efflux and synthesis was 2.16 ± 0.25 (Fig. 8). In a previous
report (20), the heat produced during the unidirectional
Ca2+ movement from the vesicle lumen to the medium was
measured by diluting vesicles previously loaded with Ca2+
in efflux media containing different concentrations of ADP,
Pi, or K+. These experiments revealed that the
Ca2+-ATPase can function in three different forms: (i) it
absorbs heat from the medium when the efflux is coupled with ATP
synthesis (
Hcal +5.01 kcal/mol of
Ca2+ released); (ii) it converts the energy derived from
the gradient into heat when Mg2+ is removed from the medium
and the synthesis of ATP is impaired, and (iii) the ATPase is no longer
able to interconvert energy when the different ligands of the enzyme
are removed from the medium, and as a result, there is no ATP synthesis
and no heat production or absorption. I now repeated these experiments,
loading the vesicles with Glc-6-P as shown in Fig. 4 and, after
centrifugation, diluting the loaded vesicles in efflux media containing
either a mixture of glucose and hexokinase or 1 mM ATP
(Table V). In agreement with the previous
report, there was heat absorption from the medium when ADP,
Pi, and Mg2+ were included in the medium, thus
favoring the synthesis of ATP (Fig. 9).
The rate of Ca2+ efflux decreased, and ATP was no longer
synthesized when Mg2+ was not added and EDTA was included
in the medium in order to chelate the small amount of Mg2+
introduced in the medium together with the Ca2+-loaded
vesicles. In this condition, the Ca2+ efflux was
exothermic, and the amount of heat released was proportional to the
amount of Ca2+ released (Fig.
10). The rate of Ca2+
efflux decreased after the addition of thapsigargin to the medium, and
in this condition there was no measurable heat release or heat
absorption during the efflux (Figs. 9 and 10 and Table V). The
Hcal values calculated using the uncoupled
efflux measured in the absence of thapsigargin varied between
13.6
and
16.3 kcal/mol of Ca2+ released. These values
decreased to
20.3 and
24.6 kcal/mol of Ca2+ when
the difference of efflux measured in the presence and absence of
thapsigargin was used to calculate the
Hcal
value (20). Knowing the
Hcal values for the
coupled and uncoupled Ca2+ efflux, it was possible to
estimate the relative contribution of the efflux and of the substrate
hydrolysis to the heat produced during steady state. In the presence of
either PEP or 1 mM ATP, most of the heat produced was
derived from the hydrolysis of ATP (Table
VI); the Ca2+ efflux
contributed with only 18.9 and 17.0%, respectively, of the
total heat released during steady state. Because most of the ATP is
cleaved through the uncoupled route (Table II), it is proposed that the
hydrolysis of ATP through reactions 2 and 10 in Fig. 2 is the catalytic
route that most contributes to the heat released during ATP hydrolysis
in Ca2+-loaded vesicles. A different pattern was observed
when Glc-6-P and Fru-1,6-P were used as the ATP-regenerating system,
where the Ca2+ efflux seems to attenuate the heat
absorption derived from the hydrolysis of the substrate needed to
maintain the gradient at steady state. Notice that in Table VI, the sum
of the coupled and uncoupled Ca2+ efflux is exothermic.
Thus, if corrected by the heat derived from the Ca2+
efflux, the amount of heat absorbed from the medium during substrate hydrolysis is +2.73 mcal (Table VI), a value well above the 0.58 and
1.09 mcal measured in Table IV.

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Fig. 8.
Ca2+ efflux and ATP
synthesis. The assay medium composition was 50 mM
MOPS-Tris buffer (pH 7.0), 2 mM MgCl2, 120 µM CaCl2, 120 µM EGTA, 50 µM ADP, 10 mM Pi, 100 mM KCl, 5 mM Fru-1,6-P, 50 µM
Fru-6-P, 10 units/ml pyruvate kinase, 5 mM
NaN3, 10 µM
P1,P5-di(adenosine-5') pentaphosphate. Medium
was divided in two samples. A trace amount of 45Ca was
added to one sample and used to measure Ca2+ uptake. To the
other sample a trace amount of 32Pi was added
after 14-min incubation as indicated by the arrow and used
to measure the synthesis of [ -32P]P. The reactions
were started by the simultaneous addition of vesicles, 40 µg of
protein/ml. After a 14-min incubation, the two media were divided into
different samples. Two of them were mixed with EGTA and Fru-6-P, final
concentrations in the medium of 5 and 20 mM, respectively.
Two other samples were mixed with the same amounts of EGTA and Fru-6-P
plus thapsigargin to a final concentration of 4 µM ( ,
). A fifth sample was not diluted and was used to measure the
Ca2+ retained by the vesicles during steady state. The
temperature was 35 °C. , Ca2+ uptake; ,
Ca2+ efflux after the addition of EGTA and Fru-6-P as
indicated by the arrow. , Ca2+ efflux with
thapsigargin; , ATP synthesis; , ATP synthesis with thapsigargin.
The calculated free Ca2+ concentration in the medium was
4.4 µM before the addition of vesicles and decreased to
0.23 µM at steady state. Shown is a representative
experiment.
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Table V
Release and absorption of heat during unidirectional Ca2+
efflux
The vesicles were loaded with Ca2+ in media containing Glc-6-P
and hexokinase as described in Fig. 4. Other additions to the assay
medium and experimental conditions were as in Figs. 9 and 10. Values
are means ± S.E. of the number of experiments (n)
shown in the table. Hk, hexokinase; TG, thapsigargin.
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Fig. 9.
Ca2+ efflux (A),
ATP synthesis (B), and heat absorption
(C). The efflux medium composition was 50 mM MOPS-Tris buffer (pH 7.0), 4 mM
MgCl2, 100 µM ADP, 10 mM
Pi, 100 mM KCl, 20 mM glucose, 10 units/ml hexokinase, 5 mM NaN3, and 10 µM P1,P5-di(adenosine-5')
pentaphosphate without thapsigargin ( ) and with 2 µM
thapsigargin ( ). The temperature was 35 °C. The vesicles were
loaded using Glc-6-P and hexokinase as shown in Fig. 4, and after
centrifugation the pellet was resuspended in a small volume of the
loading mixture and used for the simultaneous measurement of
Ca2+ efflux, ATP synthesis, and heat absorption. Other
conditions were as described under "Materials and Methods." Shown
is a typical experiment.
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Fig. 10.
Uncoupled Ca2+ efflux
(A) and heat release (B). The
assay medium and experimental conditions were the same as in Fig. 9
except that MgCl2 was omitted and 5 mM EDTA was
added to the efflux medium. , without thapsigargin; , with 2 µM thapsigargin.
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Table VI
Contribution of Ca2+ efflux and substrate hydrolysis to the
total amount of heat released or absorbed during Ca2+ transport
The heat derived from the coupled and uncoupled Ca2+ efflux was
calculated using the values of Ca2+ efflux shown in Table II
and the Hcal values of Table V. For the uncoupled
Ca2+ efflux, the Hcal value used was the
average measured with 1 mM ATP and with Glc-6-P ( 14.93
kcal/mol of Ca2+ released). The heat derived from the ATPase
was calculated by subtracting the heat derived from the total
Ca2+ efflux from the heat measured.
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DISCUSSION |
Thermogenesis--
The data presented indicate that in the
presence of a low ADP concentration, a very large fraction of the ATP
cleaved by the Ca2+-ATPase is not used to pump
Ca2+ across the reticulum membrane. When extended to the
living cell, the cleavage of ATP through reaction 10 could be
considered an apparently futile cycle without a physiological purpose.
ATP is cleaved without apparent work, and then the ADP produced is
phosphorylated by the mitochondria, leading to an increase in oxygen
consumption. The data now described suggest that the uncoupled ATPase
activity may represent an important route of heat production that
contributes to the thermogenic control of the cell. Not only
does the yield of heat produced during the hydrolysis of ATP double
(Table IV), but the ADP produced leads to an increase in the
mitochondrial respiration with more heat production. There are several
experimental reports linking the sarcoplasmic reticulum to
thermogenesis. What follows is a brief description of the
evidence. At least two different systems are known to be involved in
the process of nonshivering thermogenesis, and these are the uncoupling
proteins (UCP) and the sarcoplasmic reticulum Ca2+-ATPase
of skeletal muscle (34-38). Different UCP isoforms have already been
identified. These include UCP1, specific to brown adipose tissue; UCP2,
found in most tissues; and UCP3, which is highly expressed in skeletal
muscle. Of the three isoforms, only UCP1 is clearly involved in heat
production. The physiological role of UCP2 and UCP3 is still
controversial (35-38). The different UCP isoforms promote the
dissipation of the proton electrochemical gradient formed across the
inner mitochondrial membrane during respiration. In order to restore
the gradient and to prevent the decrease of the cytosolic ATP
concentration, the proton leakage promoted by the UCP leads to an
increase in the mitochondrial respiration rate, increase of fatty acid
oxidation, and heat production. Similar to the UCP, a high uncoupled
ATPase activity can also lead to an increase of the mitochondrial
respiration rate to maintain the cytosolic ATP concentration (34, 35).
In fact, Table II shows that in presence of PEP, the uncoupled ATPase
activity can be up to 1 order of magnitude higher than the coupled
activity needed to maintain Ca2+ inside the reticulum.
Skeletal muscle is by far the most abundant tissue of the human body
and accounts for over 50% of the total oxygen consumption in a resting
human being and up to 90% during very active muscular work.
Calorimetric measurements of rat soleus muscle indicate that 25-45%
of heat produced in resting muscle is related to Ca2+
recirculation between sarcoplasm and sarcoplasmic reticulum (39). Conditions that promote a change in the rate of heat production in
animals are usually associated with changes in expression of both the
sarcoplasmic reticulum Ca2+-ATPase and UCP proteins. Thus,
during cold adaptation, UCP1 and UCP2 but not UCP3 are overexpressed
(37, 38, 40-42). Similarly, in cold-acclimated ducklings, there is a
30-50% increase of sarcoplasmic reticulum Ca2+-ATPase,
and in these animals, 70% of the total heat production is derived from
muscle (43, 44). The expression of both sarcoplasmic reticulum
Ca2+-ATPase and UCP1 are decreased in hypothyroid rats.
In these animals, the injection of the thyroid hormone
3,5,3'-triiodo-L-thyronine increases the expression of both
Ca2+-ATPase and UCP1 (40, 45-48). A curious system that
highlights the importance of the sarcoplasmic reticulum
Ca2+-ATPase in heat production is the heater tissue of
billfishes. In marlin and swordfish, ocular muscles are transformed
into specialized heater tissues (34). During the daily fluctuations in
temperature, the swordfish reduces the temperature changes experienced
by the brain and retina by warming these tissues with the heater organ. The heater tissue is composed of modified muscle cells in which the
contractile filaments are virtually absent and the cell volume is
packed with mitochondria and a highly developed sarcoplasmic reticulum.
Energy Interconversion--
For enzymes that are able to
interconvert energy, it is generally assumed that the chemical energy
released during ATP hydrolysis is divided in two noninterchangeable
parts, one used by the enzyme to perform work and the other converted
into heat. This does not seem to be the case of the
Ca2+-ATPase. The data presented in this and previous
reports (16-20, 49) indicate that the enzyme is able to handle the
energy released during ATP hydrolysis in such a way as to modulate the
fraction used to pump Ca2+ across the membrane, the
fraction that is dissipated in the surrounding medium as heat, and the
fraction that is used to synthesize back part of the ATP cleaved. In
this view, the total amount of energy released during ATP hydrolysis is
always the same, but the enzyme would be able to regulate the
interconversion of the different forms of energy. The fraction
converted into heat during the hydrolysis of each ATP molecule varies
depending on the conditions used; it is maximal with intact vesicles
and in the absence of ADP and decreases as the ADP/ATP ratio in
the medium increases up to a point where the Ca2+ transport
is converted from an exothermic to an endothermic process (Table
IV).
The Catalytic Cycle--
During catalysis, ATP can be cleaved
through different sets of intermediary reactions, depending on whether
or not a Ca2+ gradient is formed across the vesicle
membrane. The overall reactions catalyzed by the
Ca2+-ATPase in the presence and absence of a transmembrane
Ca2+ gradient are different. This can be concluded by
comparing the sum of all the reactions of the catalytic cycle as it
flows in absence of a gradient (Fig. 1, reactions 1-6) or in the
presence of a gradient (Fig. 2, reactions 2 and 10 or reactions 1-5
and 7-9): no gradient, 2Ca2+out + ATP
ADP + Pi + 2Ca2+in; gradient, ATP
ADP + Pi.
With leaky vesicles, a part of the energy derived from ATP
hydrolysis is used to translocate Ca2+ across the membrane
(work), and a part is dissipated as heat. The low Ca2+
concentration (10 µM) available on the two sides of the
membrane is not sufficient to permit a significant binding of
Ca2+ to the enzyme forms E2-P and
E2, and as a result reactions 3 and 4 are
irreversible, the catalytic cycle flows continuously forward without
branching, and the cleavage of each ATP molecule is accompanied by the
translocation of two Ca2+ ions across the membrane.
With intact vesicles, the energy used for Ca2+
translocation is converted into osmotic energy, and after the vesicles
are filled with Ca2+, the net hydrolysis of ATP is not
associated with a net translocation of Ca2+ across the
membrane. This is clearly the case of the uncoupled ATPase activity
(reactions 2 and 10), which, according to the data of Table II, is the
major route of ATP hydrolysis at steady state in the presence of either
PEP or 1 mM ATP. After the vesicles are filled, a part of
the Ca2+ that binds to the enzyme form
E1 is pumped across the membrane (reactions 3 and 4 forward), but the high Ca2+ concentration available
inside the vesicles will promote the binding of Ca2+ to the
enzyme forms E2-P and E2.
The Ca2+ binding to E2-P promotes
the reversal of reactions 4 and 3, leading to an increase in the steady
state level of E1~P, which is then hydrolyzed
through reaction 10 in Fig. 2 (10, 15). Alternatively, the
Ca2+ that binds to the enzyme form
E2 leaves the vesicles through the uncoupled
efflux, and the energy derived from ATP hydrolysis used to translocate
Ca2+ is first converted into osmotic energy (reactions 1-5
forward), and then, as shown in Fig. 10 and Table IV, osmotic energy is
converted by the enzyme into heat (reactions 7-9). In both cases, the
net ATP hydrolysis is not coupled with a net Ca2+
translocation, and ultimately all of the energy derived from the ATP
cleaved is converted into heat.
What may appear to be difficult to understand is the notion that in
leaky vesicles part of the chemical energy released during ATP
hydrolysis is converted into work. This was analyzed in detail in early
reports when the reversal of the catalytic cycle was discovered and the
intermediary reactions of the Ca2+-ATPase catalytic cycle
were identified (2-4, 50). In short, binding energy and chemical
energy are interconverted during catalysis. The association constant
(Ka) for the binding of Ca2+ to the
enzyme form E1 is 106 M,
and the binding energy (
G0) derived from
reaction 1 in Fig. 1 is
16.9 kcal. After phosphorylation by ATP, the
enzyme undergoes a conformational change that leads to a decrease in
the enzyme affinity for Ca2+ and dissociation of the cation
on the inner side of the membrane. The Ka of
reaction 4 is 103 M, and if the
Ca2+ concentration is kept in the micromolar range on the
two sides of the membrane, then two calcium ions dissociate from the
phosphoenzyme and the
G0 of reaction 4 forward is +8.5 kcal. The sum of the
G0 of
binding and dissociation is
8.4 kcal. This value represents the free
energy needed for the translocation of two Ca2+ ions
through the membrane, and during catalysis this is provided by the
fraction of the energy derived from ATP hydrolysis that is converted
into work. This sequence is altered after formation of the gradient,
and the cleavage of ATP is no longer coupled with sequential binding
and dissociation of Ca2+ from the enzyme, as can be deduced
from the addition of the different intermediate reactions of the
catalytic cycle.
ADP Regulation and Anoxia--
A failure of mitochondrial
respiration such as noted in anoxia and ischemia promotes an increase
in the cytosolic ADP/ATP ratio. ATP is needed for several cellular
functions, and during anoxia the cell has to adapt to a lower
consumption of ATP in order to survive. A decrease in the heat
production rate is one of the earliest events noted in anoxia (51-53).
Not only will this decrease the consumption of ATP, but a drop in cell
temperature promotes a simultaneous decrease in the metabolic activity
and ATP demand of the cell. The experiments of Tables II and IV show that an increase in the ADP/ATP ratio promotes a decrease of both uncoupled ATPase activity and yield of heat generated during ATP hydrolysis. In the presence of a high ADP/ATP ratio, such as that attained when sugar phosphates are used as the ATP-regenerating system,
the Ca2+ transport is no longer exothermic but, on the
contrary, absorbs heat from the medium. Thus, when extrapolated to the
living cell, the thermogenic activity of the Ca2+-ATPase
would be directly controlled by the energetic balance of the cell.
Raising the ADP concentration in the cytosol would minimize the
contribution of the sarcoplasmic reticulum Ca2+-ATPase to
cellular thermogenesis by increasing the recovery of the ATP cleaved
(Table I) and decreasing both the rate of uncoupled ATPase activity
(Table II) and the amount of heat produced during the hydrolysis of
each ATP molecule. Conversely, a low ADP concentration, as observed in
the presence of PEP, will increase the contribution of the reticulum to
thermogenesis by favoring the uncoupled ATPase activity with more heat
production for each ATP cleaved and, at the same time, minimizing the
recovery of the ATP cleaved by the reversal of the catalytic cycle.
Heat Absorption--
This was observed in two experimental
situations, when the Ca2+ uptake was supported by Glc-6-P
and Fru-1,6-P (Fig. 6 and Table IV) and when ATP was synthesized during
active Ca2+ efflux (Fig. 9 and Table V). This finding
suggests that Ca2+-ATPase can convert thermal energy into
either osmotic or chemical energy; i.e. it is able to use
the thermal energy available in the medium to help the pumping of
Ca2+ into the vesicles (Figs. 4 and 6 and Table IV) and to
synthesize ATP from ADP and Pi (Fig. 9 and Table V). This
possibility is supported by early reports (54, 55) showing that the
Ca2+-ATPase is able to catalyze the synthesis of ATP from
ADP and Pi after a rapid temperature transition. In these
reports, synthesis was measured in the absence of a transmembrane
Ca2+ gradient and led to the conclusion that the
Ca2+-ATPase can convert thermal energy into chemical
energy. At present, we do not know the mechanism of this conversion.
Possible parameters involved are the different
G0 and
G values of the
phosphate compounds used. The
G0 values
reported in the bibliography for ATP hydrolysis vary between
7.0 and
8.0 kcal/mol, and values for PEP, Fru-1,6-P, and Glc-6-P are
14.0,
2.8, and
2.5 kcal/mol, respectively (25, 56, 57). The
G values calculated taking into account the
concentrations of reactants and products available in the medium during
steady state vary between
10.1 and
11.9 for the experiments
performed with 1 mM ATP, between
18.0 and
18.6 for PEP,
between
7.1 and
7.4 for Fru-1,6-P, and between
6.8 and
7.0 for
Glc-6-P.
Finally, the possibility that Fru-1,6-P and Glc-6-P may be used in an
ATP-regenerating system as salvage routes during anoxia has been
discussed in earlier reports (25, 26).
 |
ACKNOWLEDGEMENT |
I am grateful to Valdecir A. Suzano for
technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from PRONEX - Financiadora
de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by
Fundação de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence may be addressed. Tel.: 55-21-270-1635;
Fax: 55-21-270-8647; E-mail: demeis@bioqmed.ufjr.br.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M103318200
 |
ABBREVIATIONS |
The abbreviations used are:
PEP, phosphoenolpyruvate;
MOPS, 4-morpholinepropanesulfonic acid;
Glc-6-P, glucose 6 phosphate;
Fru-1, 6-P, fructose 1,6-diphosphate;
F-6-P, fructose 6-phosphate;
UCP, uncoupling protein(s).
 |
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