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Uncoupled ATP Hydrolysis and Thermogenic Activity of the Sarcoplasmic Reticulum Ca2+-ATPase
2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês
10.1074/jbc.m200648200
ISSN1083-351X
AutoresHosana Barata, Leopoldo de Meis,
Tópico(s)Fuel Cells and Related Materials
ResumoThe 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. 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. 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.Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar, 2.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar). This is represented in Fig. 1 as reactions 1–6flowing forward and backwards (3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar). A part of the energy released during ATP hydrolysis is not used for transport and is converted into heat. Recently (7.de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (53) Google Scholar, 8.de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar, 9.Mitidieri F. de Meis L. J. Biol. Chem. 1999; 274: 28344-28350Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.de Meis L. Biosci. Rep. 2001; 21: 113-137Crossref PubMed Scopus (51) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) 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 Me2SO 1The abbreviations used are: Me2SOdimethyl sulfoxideMOPS4-morpholinepropanesulfonic acid1The abbreviations used are: Me2SOdimethyl sulfoxideMOPS4-morpholinepropanesulfonic acid (20% v/v) is added to the medium or when the temperature of the assay medium is decreased from 35 to 20 °C (7.de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (53) Google Scholar, 8.de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar, 11.de Meis L. Biosci. Rep. 2001; 21: 113-137Crossref PubMed Scopus (51) Google Scholar). At present, we do not know why these two conditions decrease the heat production measured with Ca2+-loaded vesicles. dimethyl sulfoxide 4-morpholinepropanesulfonic acid dimethyl sulfoxide 4-morpholinepropanesulfonic acid 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.Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar, 2.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar). 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.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar,13.Inesi G. Kurzmack M. Verjovski-Almeida S. Ann. N. Y. Acad. Sci. 1978; 307: 224-227Crossref PubMed Scopus (43) Google Scholar, 14.Inesi G. Kurzmack M. Coan C. Lewis D. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). Oxalate is not available in muscles, and during transport the Ca2+ concentration inside the reticulum increased from 5 to 20 mm (4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar). When this is reproduced in vitro, the Ca2+/ATP stoichiometry measured varies between 0.3 and 0.6 (6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15.McWhirter J.M. Gould G.W. East J.M. Lee A.G. Biochem. J. 1987; 245: 731-738Crossref PubMed Scopus (17) Google Scholar, 16.Meltzer S. Berman M.C. J. Biol. Chem. 1984; 259: 4244-4253Abstract Full Text PDF PubMed Google Scholar). 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.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.de Meis L. Biosci. Rep. 2001; 21: 113-137Crossref PubMed Scopus (51) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15.McWhirter J.M. Gould G.W. East J.M. Lee A.G. Biochem. J. 1987; 245: 731-738Crossref PubMed Scopus (17) Google Scholar, 17.de Meis L. J. Biol. Chem. 1991; 266: 5736-5742Abstract Full Text PDF PubMed Google Scholar, 18.de Meis L. Wolosker H. Engelender S. Biochim. Biophys. Acta. 1996; 1275: 105-110Crossref Scopus (22) Google Scholar). 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.Yu X. Inesi G. J. Biol. Chem. 1995; 270: 4361-4367Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) 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.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 20.Fortea M.I. Soler F. Fernandez-Belda F. J. Biol. Chem. 2000; 275: 12521-12529Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 21.Logan-Smith M.J. Lockyer P.J. East J.M. Lee A.G. J. Biol. Chem. 2001; 276: 46905-46911Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) 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.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). 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.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). 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. These were derived from the longitudinal sarcoplasmic reticulum of rabbit hindleg white skeletal muscle and were prepared as previously described (22.Eletr S. Inesi G. Biochim. Biophys. Acta. 1972; 282: 174-179Crossref PubMed Scopus (304) Google Scholar). 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. This was measured by the filtration method (23.Chiesi M. Inesi G. J. Biol. Chem. 1979; 254: 10370-10377Abstract Full Text PDF PubMed Google Scholar). For 45Ca uptake, trace amounts of45Ca 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 of45Ca2+ uptake was reached. The rate of Ca2+in ⇔ Ca2+outexchange 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 of45Ca2+. These were assayed using either [γ-32P]ATP or 32Pi as previously described (24.de Meis L. Methods Enzymol. 1988; 157: 190-206Crossref PubMed Scopus (63) Google Scholar). These were measured using an OMEGA isothermal titration calorimeter from Microcal Inc. (Northampton, MA) (7.de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (53) Google Scholar, 8.de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar, 9.Mitidieri F. de Meis L. J. Biol. Chem. 1999; 274: 28344-28350Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.de Meis L. Biosci. Rep. 2001; 21: 113-137Crossref PubMed Scopus (51) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). 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 (ΔH 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. 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+outexchange, 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+outexchange, 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. 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.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar). This triggers the reversal of the catalytic cycle of the ATPase (2.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 25.Makinose M. FEBS Lett. 1971; 12: 269-270Crossref PubMed Scopus (74) Google Scholar, 26.Makinose M. Hasselbach W. FEBS Lett. 1971; 12: 267-268Crossref PubMed Scopus (109) Google Scholar) during which Ca2+ leaves the vesicles through the ATPase, and ATP is synthesized from ADP and Pi. 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).Figure 3Ca2+ uptake, Ca2+in ⇔ Ca2+outexchange (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 mmCaCl2, 0.20 mm EGTA, 10 mmPi, 100 mm KCl, and 5 mmNaN3. 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 Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IRates of ATP hydrolysis, ATP synthesis, and Ca2+in ⇔ Ca2+out exchange at steady stateConditionsnATP hydrolysis (a)ATP synthesis (b)Net ATP hydrolysis (c)Ca2+in ⇔ C2+outexchange (d)ATP hydrolysis/synthesis (a/b)Ca2+/ATP ratio (d/c)nmol/mg · min−135 °C71,422 ± 12529 ± 21,394 ± 126438 ± 4549.00.320% Me2SO (v/v) 35 °C10596 ± 38176 ± 24419 ± 55561 ± 403.41.320 °C798 ± 92 ± 196 ± 9130 ± 449.01.4The 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. Open table in a new tab 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. 2 A 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.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15.McWhirter J.M. Gould G.W. East J.M. Lee A.G. Biochem. J. 1987; 245: 731-738Crossref PubMed Scopus (17) Google Scholar, 16.Meltzer S. Berman M.C. J. Biol. Chem. 1984; 259: 4244-4253Abstract Full Text PDF PubMed Google Scholar), 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.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 25.Makinose M. FEBS Lett. 1971; 12: 269-270Crossref PubMed Scopus (74) Google Scholar,26.Makinose M. Hasselbach W. FEBS Lett. 1971; 12: 267-268Crossref PubMed Scopus (109) Google Scholar). 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.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). 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).Table IIRates of coupled and uncoupled ATPase activity and Ca2+ effluxConditionsnCa2+effluxNet Ca2+-ATPaseRatio uncoupled/coupledCoupled (a)Uncoupled (b)Coupled (c)Uncoupled (d)Efflux (b/a)ATPase (d/c)35 °C759 ± 5390 ± 57249 ± 341,137 ± 1316.64.635 °C, 20% Me2SO (v/v)10352 ± 49210 ± 58280 ± 20169 ± 400.60.620 °C73 ± 1126 ± 464 ± 235 ± 8.20.5Experimental values for calculation of coupled and uncoupled rates were from Table I. Open table in a new tab Experimental values for calculation of coupled and uncoupled rates were from Table I. 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.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar), 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.4 B and 5) or calculating the calorimetric enthalpy (ΔH cal) of ATP hydrolysis (Table III). In earlier reports (7.de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (53) Google Scholar, 8.de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar, 9.Mitidieri F. de Meis L. J. Biol. Chem. 1999; 274: 28344-28350Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.de Meis L. Biosci. Rep. 2001; 21: 113-137Crossref PubMed Scopus (51) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), 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. 4 B and5 A 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 ΔH 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 ΔH 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.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) Google Scholar) 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, Pi, 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 (ΔH 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 (ΔH cal −14.9 kcal/mol of Ca2+ released). Knowing the ΔH calvalues 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.Figure 5Heat released during ATP hydrolysis in the presence (●) and absence (○) of a Ca 2+ 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 Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIHeat released during ATP hydrolysisConditionsΔH calATPGradientLeaky vesicles35 °C−22.9 ± 1.2 (13)−12.2 ± 1.3 (16)20% Me2SO (v/v), 35 °C−10.3 ± 1.3 (10)−9.4 ± 1.3 (6)20 °C−10.5 ± 0.9 (12)−10.6 ± 1.3 (6)The assay medium composition and experimental conditions were as in Figs. 4 and 5. In the table, the numbers in parentheses indicate the number of experiments, and the values are means ± S.E. Open table in a new tab Table IVContribution of Ca2+ efflux and substrate hydrolysis to the total amount of heat releasedPreincubationHeat derived from Ca2+effluxHeat measuredHeat derived from ATPaseCoupled (a)Uncoupled (b)total (a +b)mcal/mg·min−1mcal/mg·min−1mcal/mg·min−135 °C+0.34−5.81−5.47−18.94−13.4720% Me2SO (v/v), 35 °C+2.01−2.99−0.98−4.32−3.3420 °C+0.02−1.88−1.86−1.01+0.85The heat derived from the coupled and uncoupled Ca2+ efflux was calculated using the ΔH cal values of Ca2+efflux of +5.7 and −14.9 kcal/mol of Ca2+ released, respectively (10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) Google Scholar). 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. Open table in a new tab The assay medium composition and experimental conditions were as in Figs. 4 and 5. In the table, the numbers in parentheses indicate the number of experiments, and the values are means ± S.E. The heat derived from the coupled and uncoupled Ca2+ efflux was calculated using the ΔH cal values of Ca2+efflux of +5.7 and −14.9 kcal/mol of Ca2+ released, respectively (10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) Google Scholar). 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. 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.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). 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. Earlier reports (3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar, 6.Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar) 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 E 2-P and E 2, 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 E 2-P (2.Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 3.de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (537) Google Scholar, 4.de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, New York1981: 1-163Google Scholar, 5.Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (74) Google Scholar). 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:E 1∼P (19.Yu X. Inesi G. J. Biol. Chem. 1995; 270: 4361-4367Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20.Fortea M.I. Soler F. Fernandez-Belda F. J. Biol. Chem. 2000; 275: 12521-12529Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). 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.Sumbilla C. Lewis D. Hammerschmidt T. Inesi G. J. Biol. Chem. 2002; 277: 13900-13906Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) 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.Sumbilla C. Lewis D. Hammerschmidt T. Inesi G. J. Biol. Chem. 2002; 277: 13900-13906Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the authors determine the kinetic constant of the catalytic cycle partial reactions. 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.Chinet A.E. Decrouy A. Even P.C. J. Physiol. (Lond.). 1992; 455: 663-678Crossref Scopus (42) Google Scholar, 30.Block B.A. Annu. Rev. Physiol. 1994; 56: 535-577Crossref PubMed Scopus (213) Google Scholar, 31.Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (120) Google Scholar). The data presented in this and previous reports (10.de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12.Reis M. Farange M. Souza A.C.L. de Meis L. J. Biol. Chem. 2001; 276: 42793-42800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) 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.Silva J.E. J. Clin. Invest. 2001; 108: 35-37Crossref PubMed Scopus (68) Google Scholar). 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. We are grateful to Valdecir Antunes Suzano and Antônio Carlos de Miranda for technical assistance.
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