Produção Nacional
Revisado por pares
Uncoupled ATPase Activity and Heat Production by the Sarcoplasmic Reticulum Ca2+-ATPase
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m103318200
ISSN1083-351X
Autores Tópico(s)Heat shock proteins research
ResumoSarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2+ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca2+ gradient is formed across the vesicle membrane. After Ca2+accumulation, a part of the Ca2+-ATPase activity is not coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995)J. Biol. Chem. 270, 4361–4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca2+ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca2+ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca2+ efflux is exothermic (14–16 kcal/Ca2+mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed. Sarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2+ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca2+ gradient is formed across the vesicle membrane. After Ca2+accumulation, a part of the Ca2+-ATPase activity is not coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995)J. Biol. Chem. 270, 4361–4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca2+ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca2+ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca2+ efflux is exothermic (14–16 kcal/Ca2+mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed. phosphoenolpyruvate 4-morpholinepropanesulfonic acid glucose 6 phosphate 6-P, fructose 1,6-diphosphate fructose 6-phosphate uncoupling protein(s) This work deals with two interconnected subjects: (i) the mechanism of energy interconversion by enzymes and (ii) heat generation, a process that plays a key role in the metabolic activity and energy balance of the cell. The biological preparation used was vesicles derived from the sarcoplasmic reticulum of rabbit white skeletal muscle. These vesicles retain a membrane-bound Ca2+-ATPase, which is able to interconvert different forms of energy. During Ca2+ transport, the chemical energy derived from ATP hydrolysis is used by the ATPase to pump Ca2+ across the vesicle membrane, leading to the formation of a transmembrane Ca2+ gradient (see reactions 1–6 forward in Figs. 1 and 2). In this process, chemical energy derived from ATP hydrolysis is converted into osmotic energy. After Ca2+ accumulation, the catalytic cycle of the enzyme can be reversed, and the accumulated Ca2+ leaves the vesicles through the Ca2+-ATPase synthesizing ATP from ADP and Pi (read reactions 6 to 1 backward in Figs. 1 and 2). During synthesis, osmotic energy is converted back into chemical energy (1Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 2de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (541) Google Scholar, 3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar, 4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 5Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 6Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar). In the steady state, the Ca2+ concentrations inside the vesicles and in the assay medium remain constant, but the ATPase operates simultaneously forward (ATP hydrolysis and Ca2+uptake) and backwards (Ca2+ efflux and ATP synthesis), and chemical and osmotic energy are continuously interconverted by the ATPase.Figure 2The catalytic cycle of the Ca2+-ATPase in the presence of a transmembrane Ca2+ concentration gradient. The characteristics of the enzyme forms E 1 andE 2 are the same as those of Fig. 1. This sequence is observed when the Ca2+ concentration on the outer surface of the membrane is inferior to 50 μm and the concentration in the vesicle lumen is higher than 1 mm. The high intravesicular Ca2+ concentration permits the reversing of reactions 4 and 3. As a result, the catalytic cycle can be reversed, leading to ATP synthesis and Ca2+ efflux (reactions 5 to 1 backwards). The uncoupled Ca2+ efflux is mediated by reactions 7–9 flowing forward. The uncoupled ATPase activity is mediated by reaction 10 (10–15).View Large Image Figure ViewerDownload (PPT) The catalytic cycle of the ATPase varies depending on the Ca2+ concentration in the vesicle lumen. When the free Ca2+ concentration inside the vesicles is kept in the micromolar range, the reaction cycle flows as shown in Fig. 1 (2de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (541) Google Scholar, 3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar, 4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 5Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar). The main feature of this cycle is that the hydrolysis of each ATP molecule is coupled with the translocation of two Ca2+ ions across the membrane (4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 5Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 6Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar, 7Meissner G. Biochim. Biophys. Acta. 1973; 298: 906-926Crossref PubMed Scopus (132) Google Scholar). This was best measured in pre-steady state experiments in which the luminal Ca2+ has yet to rise (8Inesi G. Kurzmack M. Verjovski-Almeida S. Ann. N. Y. Acad. Sci. U. S. A. 1978; 307: 224-227Crossref PubMed Scopus (43) Google Scholar, 9Inesi G. Kurzmack M. Coan C. Lewis D. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar, 10Yu X. Inesi G. J. Biol. Chem. 1995; 270: 4361-4367Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The enzyme cycles through two more sets of intermediary reaction when intact vesicles are used, and the Ca2+concentration inside the vesicles rises to the millimolar range (see Fig. 2). These are ramifications of the catalytic cycle and are denoted as dashed lines in Fig. 2. In one of them, a part of the Ca2+ accumulated by the vesicles leaks through the enzyme without catalyzing the synthesis of ATP. This is referred to as uncoupled Ca2+ efflux and is represented by reactions 7–9 in Fig. 2 (11Gould G.W. McWhirter J.M. Lee A.G. Biochim. Biophys. Acta. 1987; 904: 45-54Crossref PubMed Scopus (36) Google Scholar, 12de Meis L. J. Biol. Chem. 1991; 266: 5736-5742Abstract Full Text PDF PubMed Google Scholar, 13Inesi G. de Meis L. J. Biol. Chem. 1989; 264: 5929-5936Abstract Full Text PDF PubMed Google Scholar, 14de Meis L. Wolosker H. Engelender S. Biochim. Biophys. Acta. 1996; 1275: 105-110Crossref Scopus (22) Google Scholar). In 1995, Yu and Inesi (10Yu X. Inesi G. J. Biol. Chem. 1995; 270: 4361-4367Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and later Forteaet al. (15Fortea M.I. Soler F. Inesi G. J. Biol. Chem. 2000; 275: 12521-12529Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) observed that the progressive rise in the luminal Ca2+ concentrations promotes another ramification of the catalytic cycle sequence leading to ATP hydrolysis without Ca2+ translocation. According to these authors, the uncoupled ATP hydrolysis is derived from the cleavage of the phosphoenzyme form 2Ca:E 1∼P (reaction 10 in Fig. 2). In recent reports (16de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 17de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar, 18Mitidieri F. de Meis L. J. Biol. Chem. 1999; 274: 28344-28350Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19de Meis L. An. Acad. Bras. Cienc. 2000; 72: 365-379Crossref PubMed Google Scholar, 20de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) Google Scholar), it was shown that chemical and osmotic energy are not the only two forms of energy interconverted by the ATPase. During the steady state, a fraction of both chemical and osmotic energy is converted by the ATPase into heat. The total amount of energy released during ATP hydrolysis is always the same, but the fraction of the total energy that is converted into either chemical or osmotic energy or heat seems to be modulated by the ATPase. The main experimental finding that led to this conclusion was that the amount of heat released during the hydrolysis of each ATP molecule varies depending on whether or not a transmembrane gradient is formed across the vesicle membrane. In the absence of a Ca2+ gradient (leaky vesicles; see Fig. 1) between 10 and 12 kcal are released for each mol of ATP cleaved, and in the presence of a Ca2+gradient (intact vesicles; see Fig. 2) the amount of heat released increases to the range of 20–24 kcal/mol of ATP cleaved. At present, it is not clear why the amount of heat produced during the hydrolysis of each ATP molecule increases after Ca2+ accumulation. One of the catalytic routes involved in heat production seems to be the uncoupled Ca2+ efflux (20de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) Google Scholar). In this case, the energy derived from ATP hydrolysis is first converted into osmotic energy (reactions 1–4 in Fig. 2), and then during the uncoupled Ca2+ efflux (reactions 7–9), osmotic energy is converted into heat. This work now raises the possibility that the uncoupled ATP hydrolysis discovered by Yu and Inesi (10Yu X. Inesi G. J. Biol. Chem. 1995; 270: 4361-4367Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) may represent a second route of heat production. If the hydrolysis of ATP is completed before Ca2+ translocation through the membrane (reaction 10 in Fig. 2), then there is no conversion of chemical into osmotic energy, and during catalysis more chemical energy should be left available to be converted into heat. In order to test this hypothesis, I measured the rates of uncoupled Ca2+ efflux and uncoupled ATP hydrolysis in the presence of different ADP concentrations. It is known (3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar, 4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 21Ebashi S. Lipmann F. J. Cell Biol. 1962; 14: 389-400Crossref PubMed Scopus (360) Google Scholar) that reaction 2 in Fig. 2 is highly reversible (K eq ≅ 1). Therefore, during catalysis, the fraction of enzyme that accumulates in the form 2Ca:E 1∼P depends on the ratio between the ADP and ATP concentrations available in the medium. While ATP phosphorylates the enzyme form 2Ca:E 1 (reaction 2 forward), ADP drives the reversal of the reaction converting 2Ca:E 1∼P back to 2Ca:E 1. The rise in the intravesicular Ca2+ concentration promotes inhibition of the ATPase activity and an increase in the steady state level of the enzyme form 2Ca:E 1∼P. This is referred to in the bibliography as back inhibition (1Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar, 4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 5Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 6Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar), and it is the increase of 2Ca:E 1∼P level noted during the back inhibition that promotes the uncoupled ATP hydrolysis through reactions 2 and 10 in Fig. 2 (10Yu X. Inesi G. J. Biol. Chem. 1995; 270: 4361-4367Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 15Fortea M.I. Soler F. Inesi G. J. Biol. Chem. 2000; 275: 12521-12529Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Because reaction 2 is highly reversible, it should be expected that an increase of the ADP concentration in the medium should prevent the accumulation of 2Ca:E 1∼P, and if in fact the uncoupled ATP hydrolysis proceeds through reaction 10 (10, 15) and if this cleavage produces more heat than the coupled ATP hydrolysis (reactions 3–5 in Figs. 1 and 2) as I hypothesize, then both the uncoupled ATP hydrolysis and the amount of heat produced during the cleavage of each ATP molecule should decrease in the presence of a high ADP concentrations. This working hypothesis was tested in this report using different ATP-regenerating systems. These were derived from the longitudinal sarcoplasmic reticulum of rabbit hind leg white skeletal muscle and were prepared as previously described (22Eletr S. Inesi G. Biochim. Biophys. Acta. 1972; 282: 174-179Crossref PubMed Scopus (307) 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 (13Inesi G. de Meis L. J. Biol. Chem. 1989; 264: 5929-5936Abstract Full Text PDF PubMed Google Scholar, 22Eletr S. Inesi G. Biochim. Biophys. Acta. 1972; 282: 174-179Crossref PubMed Scopus (307) Google Scholar). Vesicles were preloaded with either 40Ca2+ or45Ca2+ using different assay media as described in the figure legends for Figs. 3 to 10. After 30–40 min of incubation at 35 °C, the vesicles were centrifuged at 40,000 ×g for 40 min, the supernatant was discarded, and the pellet was kept in ice and resuspended before starting the experiment in a small volume of the loading mixture to reach the final vesicle concentration of 1.0–1.5 mg of protein/ml. The vesicles loaded with45Ca2+ were used for measurement of Ca2+ efflux, and the vesicles loaded with40Ca2+ were used for calorimetric measurements and for measurement of ATP synthesis from ADP and32Pi.Figure 10Uncoupled Ca2+ efflux (A) and heat release (B). The assay medium and experimental conditions were the same as in Fig. 9except that MgCl2 was omitted and 5 mm EDTA was added to the efflux medium. ○, without thapsigargin; ●, with 2 μm thapsigargin.View Large Image Figure ViewerDownload (PPT) This was measured by the filtration method (23Chiesi M. Inesi G. J. Biol. Chem. 1979; 254: 10370-10377Abstract Full Text PDF PubMed Google Scholar). For45Ca uptake, trace amounts of 45Ca were included in the assay medium. For 45Ca efflux, vesicles previously loaded with 45Ca were used. The reaction was arrested by filtering samples of the assay medium in Millipore filters. After filtration, the filters were washed five times with 5 ml of 3 mm La(NO3)3, and the radioactivity remaining on the filters was counted using a liquid scintillation counter. For the Ca2+in ⇔ Ca2+out exchange the assay medium was divided in two samples. Trace amounts of 45Ca2+ were added to only one of the samples, and the reaction was started by the simultaneous addition of vesicles to the two media. The sample containing the radioactive Ca2+ was used to determine the incubation time where the vesicles were filled and the steady state 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 nonradioactive Ca2+. The exchange of the radioactive Ca2+ from the medium with the nonradioactive Ca2+ contained inside the vesicles was measured by filtering samples of the assay medium in Millipore filters 10, 20, 30, 40, 60, and 120 s after the addition of45Ca2+. These were assayed using either a colorimetric method or by measuring the release of 32Pi from either [γ-32P]ATP, [32P]Glc-6-P, or [1-32P]Fru-1,6-P (24de Meis L. Methods Enzymol. 1988; 157: 190-206Crossref PubMed Scopus (63) Google Scholar, 25Monteiro-Lomeli M. de Meis L. J. Biol. Chem. 1992; 267: 1829-1883PubMed Google Scholar, 26Ramos R.C.S. de Meis L. J. Neurochem. 1999; 72: 81-86Crossref PubMed Scopus (18) Google Scholar). The32Pi produced was extracted from the medium with ammonium molybdate and a mixture of isobutyl alcohol and benzene. When the colorimetric method was used, Pi was not included in the assay medium. In the various experimental conditions used, the same results were obtained with either the colorimetric method or with the use of radioactive substrate, regardless of the ATP concentrations and ATP-regenerating system used. The values of ATPase activity shown in the figures and tables are the Ca2+-dependent activity responsible for Ca2+ transport. The Mg2+-dependent activity was measured in the presence of 2 mm EGTA. The Ca2+-dependent activity was determined by subtracting the Mg2+-dependent activity from the activity measured in the presence of both Mg2+ and Ca2+. In the different experimental conditions used, the Mg2+-dependent activity represented 2–10% of the total activity measured. This was measured using32Pi as previously described (24de Meis L. Methods Enzymol. 1988; 157: 190-206Crossref PubMed Scopus (63) Google Scholar). This were measured using an OMEGA Isothermal Titration Calorimeter from Microcal Inc. (Northampton, MA) (16de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 17de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar, 18Mitidieri F. de Meis L. J. Biol. Chem. 1999; 274: 28344-28350Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19de Meis L. An. Acad. Bras. Cienc. 2000; 72: 365-379Crossref PubMed Google Scholar, 20de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) 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 35 °C, the reaction was started by injecting vesicles into the reaction cell, and the heat change during either Ca2+ uptake or Ca2+ efflux was recorded for 20–30 min. The volume of vesicle suspension injected in the cell varied between 0.02 and 0.03 ml. The heat change measured during the initial 2 min after vesicle injection was discarded in order to avoid artifacts such as the heat derived from the dilution of the medium containing the loaded vesicles into the efflux medium and the binding of ions to the Ca2+-ATPase. The duration of these events is less than 1 min. The calorimetric enthalpy (ΔH cal) was calculated by dividing the amount of heat released by the amount of either substrate hydrolyzed or Ca2+ released by the vesicles. The units used were moles for substrate hydrolyzed and Ca2+ released and kcal for the heat released. A negative value indicates that the reaction is exothermic, and a positive value indicates that it is endothermic. All experiments were performed at 35 °C. In a typical experiment, the assay medium was divided into five samples, which were used for the simultaneous measurement of Ca2+ uptake, Ca2+in ⇔ Ca2+out exchange, substrate hydrolysis, ATP synthesis, and heat release. The syringe of the calorimeter was filled with the vesicles, and the temperature difference between the syringe and the reaction cell of the calorimeter was allowed to equilibrate, a process that usually took between 8 and 12 min. After equilibration, the reaction was started by injecting the vesicles into the reaction cell. During equilibration, the vesicles used for measurements of Ca2+ uptake, Ca2+in ⇔ Ca2+out exchange, ATP hydrolysis, and ATP synthesis were kept at the same temperature, length of time, and protein dilution as the vesicles kept in the calorimeter syringe. The different reactions were started simultaneously using either empty vesicles or Ca2+-loaded vesicles. For the experiments where the unidirectional Ca2+ efflux was measured, the heat released during the efflux was corrected for the heat derived from both the binding of Ca2+ to EGTA and the heat derived from the formation of Glc-6-P from ATP and glucose as previously described (20de Meis L. Biochem. Biophys. Res. Commun. 2000; 276: 35-39Crossref PubMed Scopus (15) Google Scholar). NaN3, an inhibitor of ATP synthase, and P1,P5-di(adenosine-5′) pentaphosphate, a specific inhibitor of adenylate kinase, were added to the assay medium in order to avoid interference from possible contamination of the sarcoplasmic reticulum vesicles with these enzymes. The free Ca2+ concentration in the medium was calculated using the association constants of Schwartzenbach et al. (27Schwartzenbach G. Senn H. Anderegg G. Helvetia Chimica Acta. 1957; 40: 1886-1900Crossref Scopus (216) Google Scholar) in a computer program described by Fabiato and Fabiato (28Fabiato A. Fabiato F. J. Physiol. ( Paris ). 1979; 75: 463-505PubMed Google Scholar) and modified by Sorenson et al. (29Sorenson M.M. Coelho H.S.L. Reuben J.P. J. Membr. Biol. 1986; 90: 219-230Crossref PubMed Scopus (72) Google Scholar). A large excess of pyruvate kinase, hexokinase, or phosphofructokinase was used in order to assure that ATP was regenerated at a faster rate than it was cleaved by the Ca2+-ATPase. In control experiments, the rates of substrate hydrolysis and Ca2+ uptake were measured in the presence of different concentrations of the ATP-regenerating enzymes, and the concentration of enzyme used in all experiments described was 5–10 times higher than that needed for maximal activity (25Monteiro-Lomeli M. de Meis L. J. Biol. Chem. 1992; 267: 1829-1883PubMed Google Scholar, 26Ramos R.C.S. de Meis L. J. Neurochem. 1999; 72: 81-86Crossref PubMed Scopus (18) Google Scholar). Most of the measurements performed in this work were made after the Ca2+ uptake reached the steady state. When the vesicles are still being filled, the rate of Ca2+ uptake measured represents a balance between the Ca2+ pumped inside the vesicles by the ATPase and the rate of Ca2+ that leaves the vesicles driven by the gradient formed across the membrane. During the initial minutes of incubation, these two rates are different and cannot be measured separately. Thus, the stoichiometry between the fluxes of Ca2+ through the membrane and the rates of either ATP cleavage or ATP synthesis cannot be evaluated with precision. After the steady state is reached, the rate of efflux is the same as that of Ca2+ uptake, and by measuring the rate of Ca2+in ⇔ Ca2+outexchange it is possible to determine the value of the two rates. The exchange represents the fraction of Ca2+ that leaves the vesicles and is pumped back inside the vesicles by the ATPase. The initial velocities of Ca2+ uptake measured with 1 mm ATP and 50 μm ATP plus PEP as the ATP-regenerating system were the same (see Fig. 3A). However, when the steady state was reached, the amount of Ca2+ retained by the vesicles with PEP was larger than that accumulated with 1 mm ATP. In six experiments, the steady state levels of Ca2+ uptake were 3.90 ± 0.25 μmol/mg with PEP and 2.73 ± 0.07 μmol/mg with 1 mm ATP. These values are the mean ± S.E. The rate of Ca2+in ⇔ Ca2+outexchange measured in the presence of PEP was slower than that measured with 1 mm ATP and no ATP-regenerating system (TableI). The time course of ATP hydrolysis was found to vary depending on the condition used. With the use of PEP, the rate of hydrolysis did not vary with the incubation interval, being practically the same before and after the steady state of Ca2+ uptake was reached. With 1 mm ATP, however, a significant decrease in the ATPase activity was detected after the vesicles were filled with Ca2+ (see Fig.3B). This difference was probably related to the accumulation of ADP in the medium during the course of the reaction that drives reaction 2 in Fig. 2 backwards (1Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 2de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (541) Google Scholar, 3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar, 4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 5Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 6Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar, 13Inesi G. de Meis L. J. Biol. Chem. 1989; 264: 5929-5936Abstract Full Text PDF PubMed Google Scholar, 21Ebashi S. Lipmann F. J. Cell Biol. 1962; 14: 389-400Crossref PubMed Scopus (360) Google Scholar). The apparentK m of the enzyme form 2Ca:E 1for ATP is in the range of 1–3 μm, andK m of the form 2Ca:E 1∼P for ADP is in the range of 10–30 μm (2de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (541) Google Scholar, 3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar). In the incubation interval of 10–40 min (see Fig. 3), 15–40% of the ATP added was cleaved (i.e. the ADP concentration rose from 0.15 to 0.40 mm), being therefore sufficient to promote the reversal of reaction 2 shown in Fig. 2. In the presence of PEP, there is practically no accumulation of ADP in the medium.Table IRates of ATP hydrolysis, ATP synthesis, and Ca2+in ⇔ Ca2+out exchange at steady stateAdditionsATP hydrolysis (a)ATP synth. (b)Net ATP hydrolysis (a − b)Ca2+in⇔ Ca2+out exchangeCa2+/ATP rationmol/mg · min−150 μm ADP + 2 mmPEP826 ± 109None826 ± 109173 ± 371-aThe difference between the rates of Ca2+in ⇔ Ca2+out exchange measured in the presence of 2 mm PEP and 1 mmATP was statistically significant (p < 0.05).0.26 ± 0.06ATP, 1 mm494 ± 6050 ± 3459 ± 22285 ± 201-aThe difference between the rates of Ca2+in ⇔ Ca2+out exchange measured in the presence of 2 mm PEP and 1 mmATP was statistically significant (p < 0.05).0.65 ± 0.0850 μm ADP + 5 mm Fru-1,6-P184 ± 987 ± 10109 ± 10273 ± 372.33 ± 0.2250 μm ADP + 5 mm Glu-6-P226 ± 3936 ± 7110 ± 17237 ± 252.31 ± 0.43The assay medium composition and experimental conditions were as in Figs. 3 and 4. The Ca2+/ATP ratio was calculated by dividing the rate of Ca2+in ⇔ Ca2+out exchange by the rate of net hydrolysis of ATP. The values in the table are means ± S.E. of six experiments.1-a The difference between the rates of Ca2+in ⇔ Ca2+out exchange measured in the presence of 2 mm PEP and 1 mmATP was statistically significant (p < 0.05). Open table in a new tab The assay medium composition and experimental conditions were as in Figs. 3 and 4. The Ca2+/ATP ratio was calculated by dividing the rate of Ca2+in ⇔ Ca2+out exchange by the rate of net hydrolysis of ATP. The values in the table are means ± S.E. of six experiments. The Ca2+ concentration in the lumen of intact vesicles reaches the millimolar range in a few seconds after the transport is initiated (1Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 2de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (541) Google Scholar, 3de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. 2. John Wiley & Sons, Inc., New York1981Google Scholar, 4Tanford C. CRC Crit. Rev. Biochem. 1984; 17: 123-151Crossref PubMed Scopus (75) Google Scholar, 5Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 6Hasselbach W. Prog. Biophys. Biophys. Chem. 1964; 14: 169-222Google Scholar, 8Inesi G. Kurzmack M. Verjovski-Almeida S. Ann. N. Y. Acad. Sci. U. S. A. 1978; 307: 224-227Crossref PubMed Scopus (43) Google Scholar, 9Inesi G. Kurzmack M. Coan C. Lewis D. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). This triggers the reversal of the catalytic cycle of the ATPase (30Makinose M. FEBS Lett. 1971; 12: 269-270Crossref PubMed Scopus (75) Google Scholar, 31de Meis L. Carvalho M.G.C. Biochemistry. 1974; 13: 5032-5038Crossref PubMed Scopus (86) Google Scholar, 32Carvalho M.G.C. Souza D
Referência(s)