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J. Biol. Chem., Vol. 277, Issue 19, 16868-16872, May 10, 2002

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Uncoupled ATP Hydrolysis and Thermogenic Activity of the Sarcoplasmic Reticulum Ca²⁺-ATPase

COUPLING EFFECTS OF DIMETHYL SULFOXIDE AND LOW TEMPERATURE^*

Hosana Barata and Leopoldo de Meis

From the Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro 21941 590, Brazil

Received for publication, January 22, 2002, and in revised form, February 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The sarcoplasmic reticulum Ca²⁺-ATPase transports Ca²⁺ using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca²⁺ across the membrane, and part is dissipated as heat. At 35 °C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca²⁺ gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca²⁺ accumulation, 82% of the Ca²⁺-ATPase activity is not coupled to Ca²⁺ transport, and the ratio between Ca²⁺ transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 °C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca²⁺/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca²⁺-ATPase is responsible for both the low Ca²⁺/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vesicles derived from the sarcoplasmic reticulum of rabbit white skeletal muscle retain a membrane-bound Ca²⁺-ATPase, which is able to pump Ca²⁺ across the vesicle membrane using the chemical energy derived from ATP hydrolysis. During Ca²⁺ transport chemical and osmotic energy is interconverted by the ATPase (1, 2). This is represented in Fig. 1 as reactions 1-6 flowing forward and backwards (3-6). A part of the energy released during ATP hydrolysis is not used for transport and is converted into heat. Recently (7-12) it was found that at physiological temperature (35 °C), the heat derived from ATP hydrolysis varies depending on whether or not a Ca²⁺ gradient is formed across the vesicle membrane. With leaky vesicles (no gradient), 8-12 kcal are released during the hydrolysis of 1 mol of ATP. After formation of a Ca²⁺ gradient (intact vesicles) the heat released increased to the range of 20 to 24 kcal/mol of ATP cleaved. These findings indicate that Ca²⁺-ATPase is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel that is used for Ca²⁺ accumulation and the fraction that is dissipated as heat. The difference in heat measured in the presence and absence of a Ca²⁺ gradient is abolished when Me₂SO¹ (20% v/v) is added to the medium or when the temperature of the assay medium is decreased from 35 to 20 °C (7, 8, 11). At present, we do not know why these two conditions decrease the heat production measured with Ca²⁺-loaded vesicles.

During catalysis the hydrolysis of one ATP molecule leads to the translocation of two Ca²⁺ ions across the membrane (Fig. 1). This was measured using large amounts of vesicles, oxalate, and a small amount of Ca²⁺. In this condition, practically all of the Ca²⁺ available in the medium is rapidly stored inside the vesicles as calcium oxalate crystals, which ensures the maintenance of a low lumenal free Ca²⁺ concentration (100 µM) (1, 2). A stoichiometry of two Ca²⁺ ions transported for each ATP cleaved was also clearly demonstrated in transient kinetics experiments, where the transport was measured during the first catalytic cycle of the enzyme, i.e. before the lumenal Ca²⁺ concentration raised to a high level (4, 6, 13, 14). Oxalate is not available in muscles, and during transport the Ca²⁺ concentration inside the reticulum increased from 5 to 20 mM (4). When this is reproduced in vitro, the Ca²⁺/ATP stoichiometry measured varies between 0.3 and 0.6 (6, 10, 12, 15, 16). A high lumenal Ca²⁺ concentration leads to leakage of Ca²⁺ through the ATPase (reactions 7-9 in Fig. 1) referred to as uncoupled Ca²⁺ efflux (10-12, 15, 17, 18). In earlier reports the low values of Ca²⁺/ATP measured during transport were attributed to the leakage of Ca²⁺ from the vesicle. In 1995 Yu and Inesi (19) observed that the progressive rise of the intravesicular Ca²⁺ concentration promotes the hydrolysis of ATP without concomitant Ca²⁺ translocation through the membrane (reaction 10 in Fig. 1). This was confirmed in different laboratories (10, 12, 20, 21) and was referred to as uncoupled ATPase activity. In conditions similar to those found in the cell, the rate of the uncoupled ATPase activity is 2-8 times faster than the activity coupled to Ca²⁺ transport (10, 12). The uncoupled Ca²⁺ efflux and uncoupled ATPase activity account for the low Ca²⁺/ATP stoichiometry measured during transport. Recent reports indicate that the extra amount of heat measured during ATP hydrolysis with Ca²⁺-loaded vesicles could be derived from the uncoupled ATPase activity (10, 12).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   The catalytic cycle of the Ca2+-ATPase. The sequence includes two distinct enzymes 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. The energy of hydrolysis of the phosphoenzyme form 2Ca:E1~P is about 9 kcal higher than that of the form E2-P. When leaky vesicles are used the Ca2+ concentration (10 µM) on the two sites of the membrane does not vary during ATP hydrolysis, reaction 4 is irreversible, and the sequence flows forward from reactions 1 to 6. When intact vesicles are used, the increase in the intravesicular Ca2+ concentration leads to ramifications of the catalytic cycle, the uncoupled Ca2+ efflux mediated by reactions 7-9, and the uncoupled ATPase activity mediated by reaction 10 (3-6, 10-12, 15, 17-21).

In this report, we measured the rates of uncoupled Ca²⁺ efflux and uncoupled ATPase activity and heat production at 20 °C and at 35 °C in the presence and absence of dimethyl sulfoxide. The aim was to establish whether or not there is a correlation between the uncoupled activities of the Ca²⁺-ATPase and the differences of heat production measured in the presence and absence of a Ca²⁺ gradient. In the discussion, the relevance of these data to thermogenesis is debated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sarcoplasmic Reticulum Vesicles-- These were derived from the longitudinal sarcoplasmic reticulum of rabbit hindleg 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.

Ca²⁺ Uptake 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. 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 non-radioactive Ca²⁺. The exchange of the radioactive Ca²⁺ from the medium with the non-radioactive Ca²⁺ contained inside the vesicles was measured by filtering samples of the assay medium in Millipore filters at different intervals after the addition of ⁴⁵Ca²⁺.

ATPase Activity ATP Synthesis-- These were assayed using either [-³²P]ATP or ³²P_i as previously described (24).

Heat of Reaction-- These were measured using an OMEGA isothermal titration calorimeter from Microcal Inc. (Northampton, MA) (7-12). The calorimeter cell (1.5 ml) was filled with reaction medium, and the reference cell was filled with Milli-Q water. After equilibration at the desired temperature, the reaction was started by injecting vesicles into the reaction cell, and the heat change during ATP hydrolysis was recorded for 20-40 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 to avoid artifacts such as the heat derived from the dilution of the loaded vesicles into the reaction medium and the binding of ions to the Ca²⁺-ATPase. The duration of these events is less than 1 min. The calorimetric enthalpy (H^cal) was calculated by dividing the amount of heat released by the amount of ATP hydrolyzed. The units used were moles for substrate hydrolyzed and kcal for the heat released. A negative value indicates that the reaction is exothermic, and a positive value indicates that it is endothermic.

Experimental Methods-- In a typical experiment the assay medium was divided in 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, for the same length of time, and the same protein dilution as the vesicles kept in the calorimeter syringe. The different reactions were started simultaneously. NaN₃, an inhibitor of ATP synthase, was added to the assay medium to avoid interference from possible contamination of the sarcoplasmic reticulum vesicles with this enzyme.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ Transport, ATP Hydrolysis, and ATP Synthesis-- Both the initial velocities of Ca²⁺ uptake and the amount of Ca²⁺ retained by the vesicles were found to vary depending on the conditions used (Figs. 2 and 3). The Ca²⁺ concentration in the lumen of intact vesicles reaches the millimolar range a few seconds after the transport is initiated (2-6). This triggers the reversal of the catalytic cycle of the ATPase (2-6, 25, 26) during which Ca²⁺ leaves the vesicles through the ATPase, and ATP is synthesized from ADP and P_i. In the control experiment at 35 °C a small fraction of the ATP cleaved is synthesized back by the ATPase (Fig. 2 and Table I). At this same temperature, the addition of Me₂SO promoted both a decrease of ATP hydrolysis and a large increase of ATP synthesis rates. As a result, about 30% of the ATP cleaved during transport is synthesized back. On the other hand, there was practically no ATP synthesis at 20 °C (Table I).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Ca2+ uptake, Ca2+in  Ca2+out exchange (A), ATP hydrolysis (B), and ATP synthesis (C) at 35 °C in the presence and absence of Me2SO. The assay medium composition was 50 mM MOPS-Tris buffer (pH 7.0), 1 mM ATP, 4 mM MgCl2, 0.21 mM CaCl2, 0.20 mM EGTA, 10 mM Pi, 100 mM KCl, and 5 mM NaN3 without Me2SO (closed symbols) and with 20% (v/v) Me2SO (open symbols). The reaction was started by the addition of vesicles, 10 µg of protein/ml; A, Ca2+ uptake (, ) and Ca2+in  Ca2+out exchange (, ). The rate of Ca2+in  Ca2+out exchange was measured as described under "Experimental Procedures," and the arrow indicates the addition of trace amounts of 45Ca2+ to the tube containing vesicles loaded with non-radioactive Ca2+. The calculated free Ca2+ concentration in the media was 10.1 µM (12). The figure shows a representative experiment.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Ca2+ uptake, Ca2+in  Ca2+out exchange (A) and ATP hydrolysis (B) measured at 20 °C. The assay medium composition was 50 mM MOPS-Tris buffer (pH 7.0), 1 mM ATP, 1 mM MgCl2, 0.21 mM CaCl2, 0.20 mM EGTA, 10 mM Pi, 100 mM KCl, and 5 mM NaN3. The reaction was started by the addition of vesicles, 40 µg of protein/ml; A, Ca2+ uptake () and Ca2+in  Ca2+out exchange (). The calculated free Ca2+ concentration in the medium was 10.1 µM. The figure shows a representative experiment.

View this table: [in this window] [in a new window]   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. 2 and 3. In the table, n refers to the number of experiments, and the values are means ± S.E.

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. At 35 °C, the rate of exchange measured in the presence of Me₂SO was faster than that measured in totally aqueous medium (Fig. 2A and Table I). On the other hand, lowering the temperature from 35 to 20 °C promoted a significant decrease of the exchange rate (Table I).

Knowing the rates of Ca²⁺_in  Ca²⁺_out exchange and the rates of ATP hydrolysis and ATP synthesis at steady state it was possible to estimate the following values. (i) The net ATP hydrolysis, which represents the true amount of ATP cleaved to maintain the Ca²⁺ gradient formed across the vesicle membrane during steady state, was calculated by subtracting the rate of ATP synthesis from the rate of ATP hydrolysis (Table I). (ii) The ratio between the rates of Ca²⁺ uptake and ATP hydrolysis was calculated by dividing the rate of Ca²⁺_in  Ca²⁺_out exchange by the rate of net ATP hydrolysis at steady state (Table I). In agreement with previous reports (6, 10, 12, 15, 16), at 35 °C the value of the Ca²⁺/ATP ratio was 0.3. We now show that at steady state the ratio Ca²⁺/ATP increases from 0.3 to 1.4 and 1.3 when either the temperature is decreased to 20 °C or when Me₂SO is added to the medium, respectively. (iii) The rates of coupled and uncoupled Ca²⁺ efflux; in different laboratories it has been shown that during coupled efflux (reactions 5 to 1 flowing backwards), the release of two Ca²⁺ ions from the vesicles drives the synthesis of one ATP molecule (2-6, 10, 25, 26). 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. 1). The coupled efflux increased 6-fold after the addition of Me₂SO and was abolished at 20 °C (Table II). On the other hand, the uncoupled Ca²⁺ efflux was decreased 2-fold after the addition of Me₂SO and 3-fold at 20 °C. (iv) The rates of ATP hydrolysis coupled and uncoupled to the translocation of Ca²⁺, for the calculations of which we 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 exchange (reactions 1-5 in Fig. 1). 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. 1). At 35 °C the rate of the uncoupled ATPase activity was 4.6 times faster than the ATPase activity coupled to Ca²⁺ transport. Similar values have been found in previous reports (10, 12). We now show that there is a substantial decrease of both the uncoupled ATPase activity and the ratio between the uncoupled and coupled ATPase activity when the temperature is decreased from 35 °C to 20 °C or when Me₂SO is added to the medium (Table II).

Correlation between Heat Production, Ca²⁺ Transport and ATP Hydrolysis-- In these experiments the rate of heat release and 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 synthesized back at the different incubation intervals (net hydrolysis). Within the Ca²⁺ concentrations range used, there was no ATP synthesis when leaky vesicles were used (2-6), regardless of the temperature or the addition of Me₂SO to the medium. With intact vesicles (gradient) the rate of heat production during ATP hydrolysis and Ca²⁺ transport at 35 °C was several times faster than that measured either at 20 °C or in the presence of Me₂SO. 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 of Ca²⁺ uptake, and after that the steady state was reached. This could be visualized by plotting the heat release as a function of the amount of ATP hydrolyzed (Figs. 4B and 5) or calculating the calorimetric enthalpy (H^cal) of ATP hydrolysis (Table III). In earlier reports (7-12), it was found that at 35 °C the heat released for each ATP molecule hydrolyzed by intact vesicles (gradient) was double that measured with leaky vesicles (no gradient) and that this difference was abolished when either the temperature was decreased to 20 °C or when Me₂SO was added to the medium. This was confirmed in Figs. 4B and 5A and Table III using the same conditions as those used for the measurement of coupled and uncoupled Ca²⁺ efflux and ATP hydrolysis (Table II). The difference between the H^cal of ATP hydrolysis measured in the presence and in the absence of gradient was no longer observed when the temperature was lowered to 20 °C or when Me₂SO was added to the medium (Table III). In addition to abolish the difference of H^cal, these conditions also promoted a significant decrease of the uncoupled ATPase activity (Table II), thus suggesting that the uncoupled ATPase activity indeed contribute to the extra amount of heat produced when ATP is cleaved by the Ca²⁺-loaded vesicles. In a previous report (10, 27) 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 ADP, P_i, Mg²⁺, or K⁺. These experiments revealed that the Ca²⁺-ATPase can function at least in two different forms: (i) it absorbs heat from the medium when the efflux is coupled to ATP synthesis (H^cal +5.7 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 (H^cal 14.9 kcal/mol of Ca²⁺ released). Knowing the H^cal 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 (Table IV). The values obtained clearly indicate that at 35 °C, both in the presence and absence of Me₂SO, most of the heat produced was derived from the ATPase activity. At 20 °C the amount of heat produced was small, and it was not possible to distinguish whether the main source of heat production was the uncoupled efflux or the ATPase activity.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Heat released during ATP hydrolysis. Heat release during ATP hydrolysis was measured using assay medium and experimental conditions of Figs. 2 and 3. The values of heat produced were plotted either as a function of reaction time (A) or as a function of ATP hydrolyzed (B). , 35 °C; , 20% Me2SO at 35 °C; and , 20 °C.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Heat released during ATP hydrolysis in the presence () and absence () of a Ca2+ gradient. In A, the assay medium and experimental conditions were as in Fig. 2 at 35 °C and in B as in Fig. 3 at 20 °C. Absence of gradient refers to the addition of 10 µM ionophore A23187 to the medium.

View this table: [in this window] [in a new window]   Table IV Contribution of Ca2+ efflux and substrate hydrolysis to the total amount of heat released The heat derived from the coupled and uncoupled Ca2+ efflux was calculated using the Hcal values of Ca2+ efflux of +5.7 and 14.9 kcal/mol of Ca2+ released, respectively (10, 27). The rates of coupled and uncoupled Ca2+ efflux are from Table II, and the values of heat measured are from Table III. The heat derived from the ATPase was calculated by subtracting the heat derived from the total Ca2+ efflux from the heat measured.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Coupling of the ATPase by Me₂SO and Low Temperature-- In the two conditions there was an increase of the Ca²⁺/ATP ratio from 0.3 to 1.3 and 1.4 (Table I). This was promoted by a decrease of both the uncoupled Ca²⁺ efflux and uncoupled ATPase activity (Table II). From the two activities, the decrease of the uncoupled ATPase (7- and 32-fold) was more pronounced than that of the uncoupled efflux (2- and 3-fold). In parallel to the decrease of the uncoupled routes there was a decrease of the heat produced during ATP hydrolysis by Ca²⁺-loaded vesicles (Table III). These observations support the proposal that the ramifications of the catalytic cycle are thermogenic routes that lead to an increase of the caloric yield of ATP hydrolysis, and the values of Table IV show that the uncoupled ATPase is the route that most contributes to the increase in the caloric yield of ATP hydrolysis noted when the vesicles accumulate Ca²⁺. Although Me₂SO increased the rate of exchange (Table I), most of the Ca²⁺ leaving the vesicles was coupled to the synthesis of ATP. An interesting new finding was that Me₂SO promoted a significant decrease of the ratio between the rate of ATP hydrolysis and ATP synthesis (Table I). This ratio gives a measure of the degree of energy conservation of the system (3, 4, 10). The more that ATP is synthesized, the smaller 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.

Energy Interconversion during the Catalytic Cycle-- Earlier reports (3-6) demonstrated that during catalysis binding energy and chemical energy are interconverted by the Ca²⁺-ATPase. With leaky vesicles, 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, two Ca²⁺ ions are transported across the membrane, and the cleavage of each ATP molecule is completed with the hydrolysis of the low energy phosphoenzyme E₂-P (2-5). Thus, during catalysis, a part of the energy derived from ATP hydrolysis is used to transport Ca²⁺ across the membrane (work), and a part is dissipated as heat. This sequence is altered after formation of the gradient. The high intravesicular Ca²⁺ concentration leads to the reversal of reactions 4 and 3 and the accumulation and hydrolysis of the high energy phosphoenzyme 2Ca:E₁~P (19, 20). Consequently, the cleavage of ATP is no longer coupled to the sequential binding and dissociation of Ca²⁺ from the enzyme, there is no conversion of chemical energy into work, and more energy is available to be converted into heat.

Recently Sumbilla et al. (28) observed that the Ca²⁺/ATP coupling ratio is improved by the reduction of nucleotide concentration in the presence of the ATP regenerating system and/or complexation of lumenal Ca²⁺ with phosphate or oxalate. In this work (28), the authors determine the kinetic constant of the catalytic cycle partial reactions.

Thermogenesis-- The general interest in heat production and thermogenesis has increased during the past decade due to its implications in health and disease. Alterations of thermogenesis are noted in several diseases, such as obesity and thyroid-hormone alterations. Different studies indicate that the hydrolysis of ATP by the Ca²⁺-ATPase found in muscle sarcoplasmic reticulum is one of the heat sources contributing to the thermogenesis of animals lacking brown adipose tissue (29-31). The data presented in this and previous reports (10, 12) suggest that the uncoupled ATPase activity may represent an important route of heat production that contributes to the cell thermogenesis. In intact vesicles, the rate of the uncoupled ATPase activity is 4.6 times faster than the coupled ATPase, i.e. 82% of the total ATP cleaved by Ca²⁺-loaded vesicles is processed through the route that leads to a higher heat production (reaction 10). The uncoupled Ca²⁺ efflux also contributes with a small, but significant heat production. Table IV shows that from the total heat released during transport, 28.9% is derived from Ca²⁺ efflux and the remaining 71.1% from the ATPase activity. The heat produced in the cell is ultimately related to the turnover of ATP (32). Heat is produced during mitochondria respiration. Thus, in the living cell the Ca²⁺-ATPase would account for two sources of heat: (i) the heat produced during ATP hydrolysis and Ca²⁺ efflux and (ii) the heat derived from the increase of mitochondria activity promoted by the raise in ADP concentration generated by the ATPase activity.

	ACKNOWLEDGEMENTS

We are grateful to Valdecir Antunes Suzano and Antônio Carlos de Miranda 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 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 should be addressed. Tel.: 55-21-2270-1635; Fax: 55-21-2270-8647; E-mail: demeis@biqmed.ufrj.br

Published, JBC Papers in Press, March 5, 2002, DOI 10.1074/jbc.M200648200

	ABBREVIATIONS

The abbreviations used are: Me₂SO, dimethyl sulfoxide; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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