Correlation between Uncoupled ATP Hydrolysis and Heat Production by the Sarcoplasmic Reticulum Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase transports Ca2+ using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca2+ through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca2+gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca2+ transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca2+ transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca2+ transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca2+ dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca2+-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca2+-ATPase to modulated heat production.

The main physiological role of the sarcoplasmic reticulum Ca 2ϩ -ATPase is to remove Ca 2ϩ from the cytosol thus controlling the transition between muscle contraction and relaxation (1)(2)(3)(4). The ATPase uses the energy derived from ATP hydrolysis to transport Ca 2ϩ across the membrane thus converting chemical energy into osmotic energy. The catalytic cycle of the Ca 2ϩ -ATPase can be reversed, during which Ca 2ϩ leaves the vesicles through the ATPase, and coupled to the Ca 2ϩ efflux, ATP is synthesized from ADP and P i using the energy derived from the Ca 2ϩ gradient (3, 4 -9). Contrasting to the removal of Ca 2ϩ from the medium, there is no evidence indicating that the reversal of the Ca 2ϩ pump can play a role in muscle physiology. In recent years, two more properties of the Ca 2ϩ -ATPase were discovered that apparently are of no physiological relevance, the uncoupled Ca 2ϩ efflux (10 -12) and the uncoupled ATPase activity (13)(14)(15). During transport, part of the Ca 2ϩ accumulated by the vesicles leaks to the medium through the ATPase, but this efflux is not coupled to ATP synthesis. The uncoupled Ca 2ϩ -ATPase activity was described by Yu and Inesi (13), who observed that the progressive rise in the Ca 2ϩ concentration in the vesicle lumen promotes the hydrolysis of ATP without concomitant Ca 2ϩ translocation through the membrane. Depending on the conditions used, the rate of the uncoupled ATPase activity can be 2-to 8-folds faster than the ATPase activity coupled to Ca 2ϩ translocation (15). Uncoupled Ca 2ϩ efflux and ATPase activities represent ramifications of the catalytic cycle that are detected only when a Ca 2ϩ gradient is formed across the vesicle membrane. The cycle varies depending on the Ca 2ϩ concentration in the vesicle lumen. When the free Ca 2ϩ concentration inside and outside the vesicles is kept in the micromolar range (no gradient), the reaction cycle flows as shown in Fig. 1 (4,(7)(8)(9). The two main features of this cycle are that the hydrolysis of each ATP molecule is coupled to the translocation of two Ca 2ϩ ions across the membrane (3, 4, 6 -9) and the energy of hydrolysis (⌬G o ) of the phosphoenzyme form 2Ca:E 1 ϳP is about 9 kcal higher than that of the form E 2 -P (4, 7-9, 16, 17). The enzyme cycles through two more sets of intermediary reactions when the vesicles accumulate Ca 2ϩ and a gradient is formed across the membrane (dashed lines in Fig.  2). These correspond to uncoupled ATPase activity (reaction 10) and uncoupled Ca 2ϩ efflux (reactions [7][8][9]. Recently (18 -22) we observed that during transport, chemical and osmotic energy are 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 osmotic energy, used for the resynthesis of ATP, or is converted into heat seems to be modulated by the ATPase. The main finding leading to this conclusion is that the amount of heat released during the hydrolysis of each ATP molecule varies depending on whether or not a gradient is formed across the vesicle membrane (15, 18, 20 -22). In the absence of gradient between 10 and 12 kcal are released/mol of ATP cleaved, and in the presence of gradient the heat released increases to the range of 20 to 24 kcal/mol of ATP cleaved. This difference seems to be related to the ramifications of the catalytic cycle that arise after Ca 2ϩ accumulation (15). In one of them, the chemical energy derived from ATP hydrolysis is first converted into osmotic energy (reactions 1-4 in Fig. 2) and then, during uncoupled Ca 2ϩ efflux (reactions 7-9), osmotic energy is converted into heat (15,22). The uncoupled ATPase activity in-volves the hydrolysis of the high energy phosphoenzyme form 2Ca:E 1 ϳP, and the amount of heat released in this step is larger than that released during the hydrolysis of the low energy form E 2 -P (15). Not all the intermediary reactions catalyzed by the Ca 2ϩ -ATPase are exergonic. Calorimetric measurements revealed that heat is absorbed from the environment during the Ca 2ϩ efflux coupled to ATP synthesis (15,22), and the balance between the hydrolysis of the phosphoenzymes 2Ca:E 1 ϳP, E 2 -P and ATP synthesis seems to determine the amount of heat released during Ca 2ϩ transport and ATP hydrolysis. The observation that the Ca 2ϩ -ATPase can modulate the amount of heat released during catalysis raises the possibility that uncoupled activities and reversal of the Ca 2ϩ pump may, after all, have physiological roles related to heat generation, a process that plays a key role in thermogenesis and the metabolic control of the cell (23)(24)(25). High fluoride concentrations inhibit the Ca 2ϩ -ATPase (26 -31). When the effect of fluoride was discovered, it was not known that a large fraction of the Ca 2ϩ -ATPase activity measured with intact vesicles is not coupled to Ca 2ϩ transport nor was it known that the amount of heat produced during the hydrolysis of ATP varies depending on whether or not a Ca 2ϩ gradient is formed across the vesicles membrane. In this study, we show that fluoride inhibits the uncoupled ATPase activity and the Ca 2ϩ transport becomes tightly coupled to the hydrolysis of ATP with little heat dissipation.

MATERIALS AND METHODS
Sarcoplasmic Reticulum Vesicles-These were derived from the longitudinal sarcoplasmic reticulum (light fraction) of rabbit hind limb white skeletal muscle. They were prepared as previously described (32).
Ca 2ϩ Uptake and Ca 2ϩ in 7 Ca 2ϩ out Exchange-These were measured by the filtration method (33). For 45 Ca uptake, trace amounts of 45 Ca were included in the assay medium. The reaction was arrested by filtering samples of the assay medium through Millipore filters. After filtration, the filters were washed five times with 5 ml of 3 mM La(NO 3 ) 3 , and the radioactivity remaining on the filters was counted using a liquid scintillation counter. For Ca 2ϩ in 7 Ca 2ϩ out exchange, the assay medium was divided into two samples. Trace amount of 45 Ca 2ϩ was 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 Ca 2ϩ was used to determine the incubation time when the vesicles are filled and the steady state 45 Ca 2ϩ uptake is reached. The rate of Ca 2ϩ in 7 Ca 2ϩ out exchange was measured after steady state was reached by adding trace amount of 45 Ca 2ϩ to the second sample containing vesicles loaded with non-radioactive Ca 2ϩ . The exchange between radioactive Ca 2ϩ from the medium and the non-radioactive Ca 2ϩ contained inside the vesicles was measured by filtering samples of the assay medium through Millipore filters 5, 10, 15, 20, and 25 s after the addition of 45 Ca 2ϩ .
ATPase Activity and ATP Synthesis-This was assayed by measuring the release of 32 P i from [␥-32 P]ATP. The reaction was arrested with trichloroacetic acid, final concentration 5% (w/v). The [␥-32 P]ATP not hydrolyzed during the reaction was extracted with activated charcoal as previously described (34). Two different ATPase activities can be distinguished in sarcoplasmic reticulum vesicles (7,35,36). The Mg 2ϩ -dependent activity requires only Mg 2ϩ for its activation and is measured in the presence of 2 mM EGTA to remove contaminant Ca 2ϩ from the medium. The Ca 2ϩ -dependent ATPase activity, which is correlated with Ca 2ϩ transport, is determined by subtracting the Mg 2ϩ -dependent activity from the activity measured in the presence of both Mg 2ϩ and Ca 2ϩ . ATP synthesis was measured using 32 P i as previously described (37).
Heat of Reaction-This was measured using an OMEGA Isothermal Titration Calorimeter from Microcal, Inc. (Northampton, MA) (18, 20 -22). The calorimeter sample 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 sample cell, and the heat change was recorded for 20 min. The volume of vesicle suspension injected in the sample cell varied between 0.02 and 0.03 ml. The heat change measured during the initial 2 min after vesicles injection was discarded to avoid artifacts such as heat derived from the dilution of the vesicle suspension in the reaction medium and binding of ions to the Ca 2ϩ -ATPase. The duration of these events is less than 1 min. Calorimetric enthalpy (⌬H cal ) is calculated by dividing the amount of heat released by the amount of ATP hydrolyzed (15, 18 -22, 38). The units used are moles for substrate hydrolyzed and kilocalorie for heat released. Negative values indicate that the reaction is exothermic, and positive values indicate that it is endothermic.
Experimental Procedure-All experiments were performed at 35°C. In a typical experiment the assay media was divided in five samples, which were used for the simultaneous measurement of Ca 2ϩ uptake, Ca 2ϩ in 7 Ca 2ϩ out exchange, substrate hydrolysis, ATP synthesis, and heat release. The syringe of the calorimeter was filled with 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. During equilibration, the vesicles used for measurements of Ca 2ϩ uptake, Ca 2ϩ in 7 Ca 2ϩ 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. These different measurements were started simultaneously with vesicles to a final concentration of 0.01 mg/ml. NaN 3 (5 mM), an inhibitor of ATP synthase, was added to the assay medium to avoid FIG. 1. The catalytic cycle of the Ca 2؉ -ATPase in the absence of a Ca 2؉ gradient. The sequence includes two distinct enzyme conformations, E 1 and E 2 . The Ca 2ϩ binding sites in the E 1 conformation face the external surface of the vesicle and have high affinity for Ca 2ϩ (K a ϭ 10 Ϫ6 M at pH 7.0). In the E 2 conformation the Ca 2ϩ binding sites are faced to the vesicle lumen and have low affinity for Ca 2ϩ (K a ϭ 10 Ϫ3 M at pH 7.0). In the E 1 conformation the enzyme may be phosphorylated by ATP but not P i , and conversely, E 2 is phosphorylated by P i but not by ATP. When the Ca 2ϩ concentration inside and outside the vesicles is lower than 50 M, reaction 4 is irreversible and this drives the sequence forward through reactions 1-6. For further details see Refs. 4, 7, and 8.
FIG. 2. The catalytic cycle of the Ca 2؉ -ATPase in the presence of a Ca 2؉ gradient. The characteristics of the enzyme forms E 1 and E 2 are the same as those described in Fig. 1. This sequence is observed when the Ca 2ϩ concentration on the outer surface of the vesicles is lower than 50 M and inside the vesicles is higher than 1 mM. The high intravesicular Ca 2ϩ concentration favors the reversion of reactions 4 and 3. As a consequence, the catalytic cycle can be reversed leading to Ca 2ϩ efflux and ATP synthesis (steps 5-1, backward). Uncoupled Ca 2ϩ efflux occurs through reactions 7-9 in the forward direction (12). Uncoupled ATPase activity is processed through reaction 10 (13-15).
interference from possible contamination of the sarcoplasmic reticulum vesicles with this enzyme. The free Ca 2ϩ concentration in the medium was calculated using the association constants of Schwartzenbach et al. (39) in a computer program described by Fabiato and Fabiato (40) and modified by Sorenson et al. (41).

Effects of Fluoride on the Rates of Ca 2ϩ Uptake and ATP
Hydrolysis-In agreement with previous reports (27,28,30,31), we observed that fluoride inhibits the sarcoplasmic reticulum Ca 2ϩ -ATPase (Figs. 3A and 4). Now, we show that contrasting to the inhibition of the ATPase activity, both the initial rate of Ca 2ϩ uptake and the maximal amount of Ca 2ϩ accumulated by the vesicles are enhanced by fluoride (Figs. 3B, 4, and Table I). The maximal effect is obtained in presence of 20 mM NaF (Fig. 4). The inhibition of the ATPase activity increases as the vesicles are filled with Ca 2ϩ , and maximal inhibition is observed after the steady state Ca 2ϩ uptake is reached (Table  I). These combined effects of fluoride on the ATPase activity and Ca 2ϩ uptake indicate that in the presence of fluoride the vesicles are able to accumulate more Ca 2ϩ with less ATP hydrolysis (Table I). In fact, the ratio between the initial rates of Ca 2ϩ uptake and ATP hydrolysis is 0.26 Ϯ 0.06 (5) in the absence of NaF and increases to 1.63 Ϯ 0.13 (7) in the presence of 20 mM NaF. These values are mean Ϯ S.E. of the number of experiments shown in parenthesis. Notice in Fig. 3A that the inhibition of the ATPase activity becomes more pronounced after 5 min incubation. Nevertheless, the vesicles are able to pump Ca 2ϩ even after prolonged incubation intervals with NaF. This was shown by diluting the vesicles in a medium containing 20 mM NaF and adding small amounts of Ca 2ϩ at different incubation intervals (Fig. 5). It was found that the Ca 2ϩ added is rapidly taken up by the vesicles even after 20 min of incubation (Fig. 3A).
The uncoupled ATPase activity described by Yu and Inesi (13) is better detected after the vesicles are filled with calcium (15). A possible explanation to the effects of NaF noted in Figs. Table I is that fluoride inhibits the uncoupled Ca 2ϩ -ATPase activity to an extent larger than the ATPase activity actually responsible for Ca 2ϩ uptake (reactions 1-6 in Fig. 2). This could be confirmed in experiments in which the vesicles are preincubated with NaF, and then the concentration of fluoride in the assay medium is decreased to a very low level.

3-5 and
Preincubation with Fluoride-Earlier studies indicate that fluoride binds tightly to the enzyme form E 2 but not to the form 2Ca:E 1 (27,31,42). The enzyme-fluoride complex formed in the absence of Ca 2ϩ is very stable and can be isolated free of other components by 48 h of dialysis at 4°C (27). This protective effect of Ca 2ϩ is confirmed in Fig. 6 where vesicles were preincubated without ATP and with 20 mM NaF either in presence or absence of Ca 2ϩ . After different intervals, samples of the mixture were diluted 75-fold in the assay media containing ATP used to measure the enzyme activities. The presence of Ca 2ϩ during the preincubation period prevented the inhibition of the Ca 2ϩ -ATPase by fluoride. When Ca 2ϩ is substituted by EGTA, both the rate of Ca 2ϩ uptake and the maximal amount of Ca 2ϩ accumulated by the vesicles are diminished by fluoride (Fig. 7A). However, the ATPase activity is inhibited to a far larger extent than Ca 2ϩ uptake. Both control vesicles and vesicles preincubated with NaF are able to resynthesize part of the ATP cleaved (Fig. 7). The values of net ATP hydrolysis shown on Tables II and III represent the true amount of ATP cleaved during Ca 2ϩ transport and are calculated by subtracting the rate of ATP synthesis from the rate of ATP hydrolysis. For control vesicles, the ratio between the initial rate of Ca 2ϩ uptake and net ATP hydrolysis varies between 0.68 (Table II) and 0.33 (Table III), values similar to those described in the bibliography (43). We now show that after fluoride treatment, this ratio raises to values close to 2, the optimal stoichiometric value for Ca 2ϩ transport. Taken together, these data indicate that after 20 min of preincubation with EGTA and fluoride the vesicles accumulate Ca 2ϩ at a slower rate than the control vesicles, but the transport is optimized with low energy dissipation, i.e. less ATP cleavage is needed to fill the vesicles with Ca 2ϩ (Table II) and, after steady state, to maintain the gradient (Table III).
The difference between the initial rates and maximal amount of Ca 2ϩ uptake noted in Figs. 3B and 7A are probably related to the amount of the NaF available in the assay medium. In Fig. 3B the NaF concentration varies from 5 to 20 mM, while in Fig. 7, after dilution of the preincubation mixtures, the NaF concentration is 0.27 mM for both control and NaF-preincubated vesicles. Fluoride diffuses through the membrane, and, similar to phosphate and oxalate (44), complexes with calcium acting as a calcium-precipitating agent thus decrease the lumenal free Ca 2ϩ concentration and increase the loading capacity of the vesicles. In fact, in the absence of either P i or oxalate, 20 mM fluoride greatly increased the loading capacity of the vesicles (data not shown). Thus, under the conditions of Fig. 7, we measured only the effect derived from the binding of fluoride to the enzyme. However, in Fig. 3 fluoride has two effects: it binds to the enzyme and acts as a Ca 2ϩ -precipitating agent increasing the loading capacity of the vesicles.
Ca 2ϩ Dependence and Effects of Thapsigargin and Ca 2ϩ Ionophore-The slow ATPase activity detected in intact vesicles preincubated with NaF and EGTA has the same Ca 2ϩ dependence as the control vesicles. The K value for Ca 2ϩ is 0.34 M, both for control and vesicles preincubated with NaF (Fig.  8). The Ca 2ϩ -dependent ATPase activity of intact vesicles preincubated with NaF was inhibited by thapsigargin, a specific inhibitor of the sarcoplasmic or endoplasmic reticulum Ca 2ϩ -ATPase ATPases (data not shown). The effect of fluoride varies depending on the membrane permeability for Ca 2ϩ . There is no measurable Ca 2ϩ -dependent ATPase activity when vesicles preincubated with 20 mM NaF and EGTA are diluted in an assay medium containing the Ca 2ϩ ionophore A23187. In seven different measurements the total ATPase activity measured in the presence of Ca 2ϩ plus Mg 2ϩ and the Mg 2ϩ -dependent activity measured in the presence of Mg 2ϩ and 2 mM EGTA were 1.32 Ϯ 0.31 and 1.10 Ϯ 0.21 mol P i /mg 15 min Ϫ1 , respectively. This difference is not statistically significant (p Ͼ 0.1).
ATP Synthesis-The Ca 2ϩ concentration in the lumen of intact vesicles reaches the millimolar range a few seconds after the transport is initiated (3,4,(7)(8)(9), and this triggers the reversal of the catalytic cycle of the ATPase (3, 6, 45-47) during which ATP is synthesized from ADP and P i . Fluoride inhibited the synthesis of ATP, but this inhibition was smaller than that of ATP hydrolysis (compare Fig. 7, B and C). During the initial incubation intervals, the rate of ATP synthesis by control vesicles is about 2-folds faster than that of vesicles pretreated with fluoride ( Fig. 7C and Tables II and III). This difference decreases when the vesicles are filled with Ca 2ϩ and the steady state is reached. The ratio between the rates of ATP hydrolysis and ATP synthesis (Tables II and III) is one of the parameters used to measure the degree of energy conservation of this system (7,46,47). The smaller this ratio, the more energy is being conserved by the system, i.e. the steady state can lasts longer because the net decline of the ATP concentration in the medium proceeds at a slower rate. The vesicles preincubated with fluoride are able to conserve more energy than the control vesicles because they are able to synthesize back a larger fraction of the ATP cleaved than the control vesicles both during the initial incubation intervals (Table II) and after the steady state is reached (Table III).
Ca 2ϩ in 7 Ca 2ϩ out Exchange and Rates of Coupled and Uncoupled Ca 2ϩ -ATPase-These measurements were made after the Ca 2ϩ uptake reached the steady state. When the vesicles are still being filled, the rate of Ca 2ϩ uptake measured represents a balance between the Ca 2ϩ pumped inwardly by the ATPase and the Ca 2ϩ 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 Ca 2ϩ through the membrane and the rates of either ATP cleavage or ATP synthesis cannot be evaluated precisely. After steady state is reached, the rate of efflux is the same as that of Ca 2ϩ uptake, and by measuring the rate of Ca 2ϩ in 7 Ca 2ϩ out exchange, it is possible to determine the values of these two rates. The exchange represents the fraction of Ca 2ϩ that leaves the vesicles and is pumped back inward by the ATPase. Contrasting to the effect of fluoride on both Ca 2ϩ uptake and ATP hydrolysis, the rate of Ca 2ϩ in 7 Ca 2ϩ out exchange measured at the steady state is practically not modified by fluoride pretreatment (Fig. 7A and Table III). Using the values of Ca 2ϩ in 7 Ca 2ϩ out exchange it is possible to estimate the rates of coupled and uncoupled Ca 2ϩ efflux (Table IV). In different laboratories it has already been shown that the release of two Ca 2ϩ ions from the vesicles drives the synthesis of one ATP molecule (3, 4, 6 -9, 48). The coupled Ca 2ϩ efflux (reactions 5-1 in Fig. 2) is therefore calculated by multiplying the rate of ATP synthesis by 2, and the difference between the rate of Ca 2ϩ in 7 Ca 2ϩ out exchange and the coupled Ca 2ϩ efflux represents the uncoupled Ca 2ϩ efflux (reactions 7-9 in Fig. 2). Following the same rationale it is possible to calculate the rates of ATP hydrolysis coupled and uncoupled to the translocation of Ca 2ϩ (Table IV), assuming a value of two Ca 2ϩ ions pumped for each ATP molecule cleaved. Thus, the rate of Ca 2ϩ in 7 Ca 2ϩ out exchange shown on Table III divided by 2 gives us the rate of coupled ATP hydrolysis, i.e. the ATP cleaved to pump back the Ca 2ϩ that leaves the vesicles (reaction 1-5 in Fig. 2). The difference between the total Ca 2ϩ -dependent ATP hydrolysis measured and the coupled ATP hydrolysis gives the value of the uncoupled ATPase activity (reactions 2 and 10 in Fig. 2).  The data on Table IV show that pretreatment of the vesicles with NaF promotes a small increase of the uncoupled Ca 2ϩ efflux and strongly inhibits the uncoupled ATPase activity. The coupled ATPase activity is the same for control and pretreated vesicles; however, control vesicles hydrolyzed five times more ATP than that needed to pump back the Ca 2ϩ that leaks from the vesicles (uncoupled ATPase) while the NaF-treated vesicles hydrolyzed only the ATP needed to maintain Ca 2ϩ inside the vesicles. This result suggests that at steady state, fluoride inhibits preferentially the uncoupled ATPase activity. This explains why for fluoride pretreated vesicles the ratio between Ca 2ϩ pumped by the vesicles and the rate of ATP hydrolysis is close to the optimal value 2 (Tables II and III).
Heat Production in Presence and Absence of Ca 2ϩ Gradient-The Mg 2ϩ -dependent ATPase activity measured in the presence of Mg 2ϩ and excess EGTA is not modified by 20 mM NaF. Both the rates of hydrolysis and heat released by vesicles preincubated in the presence or absence of NaF are the same ( Fig. 9 and Table V). In these experiments the values of heat released/mol of ATP hydrolyzed (⌬H cal ) found are the same as those previously measured for the Mg 2ϩ -dependent ATPase activities of vesicles derived from either skeletal muscle or blood platelets (20,21). In the presence of Mg 2ϩ and Ca 2ϩ , the addition of NaF promoted a drastic decrease of both the rates of ATP hydrolysis and heat release. The decrease in heat production, however, is more pronounced than that in the ATPase activity, thus, for the Ca 2ϩ -dependent ATPase activity, the amount of heat released for each mole of ATP cleaved (⌬H cal ) by vesicles pretreated with NaF is six to seven times smaller than that measured with control vesicles. This is observed either when NaF is added directly to the assay (Fig. 10) or when the vesicles are preincubated with NaF ( Fig. 11 and Table V).
In previous reports (15, 18 -22) it has been shown that the collapse of the Ca 2ϩ gradient leads to an increase of the Ca 2ϩdependent ATPase activity without concomitant increase in heat release, and as a result, the yield of heat produced during the hydrolysis of each ATP molecule in the presence of a gradient is 2-fold larger than that measured in the presence of the Ca 2ϩ ionophore A23187 (leaky vesicles). This is confirmed in Figs. 10 and 11 and on Table V. As shown above, there is no measurable Ca 2ϩ -dependent ATPase activity when vesicles preincubated with EGTA and NaF are diluted in media containing A23187, i.e. the amount of heat released and ⌬H cal are those determined by the Mg 2ϩ -dependent ATPase. Therefore, with NaF it is not possible to compare the values of ⌬H cal with and without gradient.  The difference between the value was statistically significant with p Ͻ 0.001. b The difference between the value was statistically significant with p Ͻ 0.001. c The difference between the value was statistically significant with p Ͻ 0.001. d The difference between the value was statistically significant with p Ͻ 0.02.

TABLE III Vesicles preincubated with 20 mM fluoride and rates of Ca 2ϩ
in 7 Ca 2ϩ out exchange, ATPase activity and ATP synthesis at steady state Preincubation and activities were measured as described for Fig. 6  The difference between the value was statistically significant with p Ͻ 0.01. b The difference between the value was statistically significant with p Ͻ 0.01. c The difference between the value was statistically significant with p Ͻ 0.001.
Energy Balance-In previous work (15,22), the heat produced during the unidirectional Ca 2ϩ movement from the vesicle lumen to the medium by diluting vesicles previously loaded with Ca 2ϩ into efflux media containing different concentrations of ADP, P i , Mg 2ϩ , or K ϩ has been measured. These experiments revealed that the Ca 2ϩ -ATPase can work in at least two different forms: i) it absorbs heat from the medium when the efflux is coupled to ATP synthesis (⌬H cal ϩ 5.7 kcal/mol Ca 2ϩ released) and ii) it converts the energy derived from the gradient into heat when Mg 2ϩ is removed from the medium and the synthesis of ATP is impaired. In such a condition, the Ca 2ϩ efflux is exothermic (⌬H cal Ϫ 14.9 kcal/mol Ca 2ϩ released). Knowing the ⌬H cal values for coupled and uncoupled Ca 2ϩ efflux, it is possible to estimate the relative contributions of the efflux and that of substrate hydrolysis to the heat produced during steady state either in the presence or absence of fluoride (Table VI). Under both conditions, the amount of heat produced by the Ca 2ϩ efflux is small. In the absence of fluoride (control) all the heat produced is derived from the hydrolysis of ATP, and in this condition most of the ATP is cleaved through the un-  Fig. 7. For the total ATPase activity (q, OE), the assay medium composition was 50 mM MOPS-Tris buffer (pH 7.0), 4 mM MgCl 2 , 0.20 mM EGTA, 10 mM P i , 100 mM KCl, 5 mM NaN 3 , 1 mM ATP, and different CaCl 2 concentrations to achieve the different free Ca 2ϩ concentrations shown in the figure. The assay media for the Mg 2ϩ -dependent activity (*) was the same as that used for the total ATPase but without CaCl 2 , and the EGTA concentration was raised to 2 mM. The Ca 2ϩ -dependent ATPase activity (E, ‚) was calculated subtracting the Mg 2ϩ -dependent from the total activity ATPase activity. Values are mean Ϯ S.E. of three experiments.  coupled route (Table IV). Therefore, the hydrolysis of ATP through reactions 2 and 10 in Fig. 2 is probably the catalytic route that mostly contributes to the heat released during ATP hydrolysis in the control experiments. The low heat production noted after fluoride treatment is probably derived from the uncoupled Ca 2ϩ efflux.

DISCUSSION
Correlation with the Bibliography-Most of the earlier experiments with NaF were performed using leaky vesicles, a condition in which the inhibitory activity of fluoride is enhanced and the recovery of the Ca 2ϩ -ATPase activity after removal of fluoride from the medium is unfavorable (27,28,30). Murphy and Coll (28) first described that the effect of fluoride is prevented by the binding of Ca 2ϩ to the high affinity binding sites of the enzyme form E 1 . This is confirmed in Fig. 6. Using vesicles solubilized with the detergent C12E9 (poly(oxyethylene)9-lauryl ether) Murphy and Coll (27) noted that the activity of fluoride-pretreated vesicles is more rapidly recovered after the excess fluoride is removed from the medium by 48 h of dialysis in the presence of millimolar Ca 2ϩ concentration. Under these conditions, the reversal of fluoride inhibition by high Ca 2ϩ concentration is very slow, with a t 1/2 of 16 h at 37°C. We observed that partial protection of the coupled ATPase activity is obtained provided that the integrity of the vesicle membrane is preserved. This protection is specific for the coupled ATPase, the uncoupled ATPase being completely inhibited by fluoride regardless of the membrane integrity. Notice in Fig. 7 that after dilution of vesicles preincubated with NaF, there is a lag phase before the vesicles are able to accumulate Ca 2ϩ . This was consistently noted in all experiments performed. At present we do not know the cause. One possibility is that during the initial incubation interval the vesicles accumulate Ca 2ϩ at a very slow rate and the progressive rise in the internal Ca 2ϩ concentration propitiates the activation of the coupled ATPase activity thus enhancing the rate of Ca 2ϩ uptake.
Coupling Ratio between Ca 2ϩ Uptake and ATP Hydrolysis-In the bibliography, this stoichiometry has been proved to be difficult to measure. A value of 2 was deduced from experiments measuring the binding of Ca 2ϩ to the enzyme in the absence of ATP and from transient kinetics measurements in which the Ca 2ϩ accumulated by the vesicles is determined in the initial milliseconds of reaction, i.e. before the Ca 2ϩ concentration in the vesicles lumen reaches the millimolar range (9,43,49). Another experimental approach used to determine the stoichiometry has been to measure the reversal of the Ca 2ϩ pump, in which the amount of ATP synthesized is correlated  11. Total ATPase activity (A), heat production (B), and ⌬H cal (C) of vesicles preincubated with NaF. Preincubation without and with 20 mM NaF and assay media were as described in Fig.  8 for the total ATPase activity. (E) Control and (q) vesicles were preincubated with 20 mM NaF. The figure shows a typical experiment and the inset in B shows the heat production with 20 mM NaF plotted in an enhanced scale. with the rate of Ca 2ϩ efflux (4,6,15). The Ca 2ϩ concentration inside the vesicles raises to the millimolar range after one or two catalytic cycles of the enzyme are completed (43). The Ca 2ϩ -ATP ratio measured after a few seconds of incubation is smaller than 2, and this has been attributed to Ca 2ϩ leakage through the membrane and back fluxes of Ca 2ϩ through the ATPase protein itself. The recent discovery of the uncoupled ATPase activity indicates that the low Ca 2ϩ -ATP values usually measured are not due to Ca 2ϩ leakage but rather to a dissociation between the catalytic and transport functions of the ATPase. Recently (15) it has been shown that the uncoupled ATPase is impaired when the ADP concentration in the medium is higher than that of ATP. In this condition, a Ca 2ϩ -ATP ratio of approximately 2 is measured after the steady state is reached. In this report it is shown that the uncoupled ATPase activity can be selectively impaired by fluoride with discrete effects on the coupled ATPase. As a result, the amount of ATP cleaved is practically only that necessary to pump Ca 2ϩ , and a Ca 2ϩ -ATP ratio near 2 can be measured both during the initial incubation intervals and after the steady state is reached and the vesicles are filled with Ca 2ϩ . The identical Ca 2ϩ dependence of the activities inhibited more easily and less easily by fluoride demonstrate unambiguously that the uncoupled activity is an actual pathway of the native Ca 2ϩ -ATPase, rather than due to partial denaturation or a contaminating phosphatase.
Energy Interconversion-The data presented in this and previous reports (15,18,20,21) indicate that the enzyme is able to determine the fate of the energy released during ATP hydrolysis in such a way as to modulate the fraction used to pump Ca 2ϩ across the membrane, the fraction that is dissipated to 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 these different forms of energy. In this work it is shown that the amount of heat released during ATP hydrolysis by the Ca 2ϩ -ATPase varies depending on whether or not the vesicles are preincubated with fluoride (Table V). With intact vesicles and in the absence of fluoride, 20 -22 kcal are released during the hydrolysis of each ATP molecule, and after fluoride pretreatment, only 3 kcal/mol of ATP cleaved are released. The different values found with and without fluoride indicate that when no work is being performed (uncoupled ATPase), most of the energy is dissipated as heat. With fluoride practically all the ATPase activity is coupled to Ca 2ϩ transport, and therefore most of the energy is converted into work and little is dissipated as heat (coupled ATPase). Furthermore, the relationship between the rates of ATP hydrolysis and ATP synthesis is found to decrease about 3-fold after NaF treatment (Table III), indicating that the abolishment of the uncoupled ATPase activity by fluoride leads to an increase of the degree of energy conservation by the system. Finally, the possible physiological implications of the thermogenic activity of the Ca 2ϩ -ATPase has been discussed in a recent report (15).