«UNCOUPLED ATPase ACTIVITY AND HEAT PRODUCTION BY THE SARCOPLASMIC RETICULUM Ca2+-ATPase: REGULATION BY ADP * Leopoldo de Meis From the Instituto de ...»
JBC Papers in Press. Published on May 7, 2001 as Manuscript M103318200
UNCOUPLED ATPase ACTIVITY AND HEAT PRODUCTION BY THE
SARCOPLASMIC RETICULUM Ca2+-ATPase: REGULATION BY ADP *
Leopoldo de Meis
From the Instituto de Ciências Biomédicas, Departamento de Bioquímica Médica,
Universidade Federal do Rio de Janeiro, Cidade Universitária, RJ 21941-590,
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Running title: Heat production by the Ca2+-ATPase Address correspondence to Leopoldo de Meis, Tel: int + 55 21 270-1635, Fax int + 55 21 270-8647, E-mail firstname.lastname@example.org Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
INTRODUCTIONThis 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
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 backwards in Figs. 1 and 2). During synthesis, osmotic energy is converted back into chemical energy (1-6). In the steady state the Ca2+ concentrations inside the vesicles and in the assay medium remain constant but the ATPase operates simultaneously forward (ATP hydrolysis and Ca2+ uptake), and backwards (Ca2+ efflux and ATP synthesis) and chemical and osmotic energy are continuously interconverted by the ATPase.
The catalytic cycle of the ATPase varies depending on the Ca2+ concentration in the vesicle lumen. When the free Ca2+ concentration inside the vesicles is kept in the micromolar range, the reaction cycle flows as shown in Fig. 1 (2- 5). The main feature of this cycle is that the hydrolysis of each ATP molecule is coupled with the translocation of two Ca2+ ions across the membrane (4-7). This was best measured in pre-steady state experiments in which the lumenal Ca2+ has yet to rise (8-10). The enzyme cycles through two more sets of intermediary reaction when intact vesicles are used, and the Ca2+ concentration inside the vesicles rises to the millimolar range (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, 8 and 9 in Fig.2 (11-14). In 1995 Yu and Inesi (10) and later Fortea et al (15) observed that the progressive rise in the lumenal Ca2+
leading to ATP hydrolysis without Ca2+ translocation. According to these authors, the uncoupled ATP hydrolysis is derived from the cleavage of the phosphoenzyme form 2Ca:E1~P (reaction 10 in Fig. 2).
In recent reports (16-20) it was shown that chemical and osmotic energy are not the only two forms of energy interconverted by the ATPase. During the steady state, a fraction of both chemical and osmotic energy is converted by the ATPase into heat. The total amount of energy released during ATP hydrolysis is always the same, but the fraction of the total energy that is converted into either chemical or osmotic energy or heat seems to be modulated by the ATPase. The main experimental finding that led to this conclusion was that the amount of heat released during the hydrolysis of each ATP molecule varies depending on whether or not a transmembrane gradient is formed across the vesicle membrane. In the absence of a Ca2+ gradient (leaky vesicles, 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, Fig. 2) the amount of heat released increases to the range of 20 to 24 kcal for each mole of ATP cleaved. At present it is not clear why the amount of heat produced during the hydrolysis of each ATP molecule increases after Ca2+ accumulation. One of the catalytic routes involved in heat production seems to be the uncoupled Ca2+ efflux (20). In this case, the energy derived from ATP hydrolysis is first converted into osmotic energy (reactions 1 to 4 in Fig. 2) and then during the uncoupled Ca2+ efflux (reactions 7 to 9), osmotic energy is converted into heat. We now raise the possibility that the uncoupled ATP hydrolysis
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 we measured the rates of uncoupled Ca2+ efflux and uncoupled ATP hydrolysis in the presence of different ADP concentrations. It is known (3, 4, 21) that reaction 2 in Fig. 2 is highly reversible (Keq ≅ 1). Therefore, during catalysis the fraction of enzyme that accumulates in the form 2Ca:E1~P depends on the ratio between the ADP and ATP concentrations available in the medium. While ATP phosphorylates the enzyme form 2Ca:E1 (reaction 2 forward), ADP drives the reversal of the reaction converting 2Ca:E1~P back to 2Ca:E1. The rise in the intravesicular Ca2+ concentration promotes inhibition of the ATPase activity and an increase in the steady state level of the enzyme form 2Ca:E1~P. This is referred to in the bibliography as back inhibition (1, 3 - 6) and it is the increase of 2Ca:E1~P level noted during the back inhibition that promotes the uncoupled ATP hydrolysis through reactions 2 and 10 in Fig. 2 (10, 15). Because reaction 2 is highly reversible, it should be expected that an increase of the ADP concentration in the medium should prevent the accumulation of 2Ca:E1~P, and if in fact the uncoupled ATP hydrolysis proceeds through reaction 10 (10, 15), and if this cleavage produces more heat than the coupled ATP hydrolysis (reactions 3 to 5 in Figs.1 and 2) as we hypothesize, then both the uncoupled ATP hydrolysis and the amount of heat produced during the cleavage of each ATP molecule should
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 a significant amounts of ryanodine-sensitive Ca2+ channels. The vesicles also did not exhibit the phenomenon of Ca2+-induced
incubation at 35oC, 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
were used for calorimetric measurements and for measurement of ATP synthesis from ADP and 32Pi.
Ca2+ uptake, Ca2+ efflux and Ca2+in⇔Ca2+out exchange. This was measured by
used. The reaction was arrested by filtering samples of the assay medium in Millipore filters. After filtration, the filters were washed five times with 5ml of 3mM La(NO3)3 and the radioactivity remaining on the filters was counted using a liquid
the vesicles was measured by filtering samples of the assay medium in Millipore filters 10, 20, 30, 40, 60 and 120 sec after the addition of 45Ca2+.
ATPase activity, cleavage of PEP 1, gluc.6-P and fruct. 1, 6-P. These were assayed using either a colorimetric method or by measuring the release of Pi
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 2mM 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% to 10% of the total activity measured.
ATP synthesis. This was measured using 32Pi as previously described (24).
Heat of reaction. These were measured using an OMEGA Isothermal Titration Calorimeter from Microcal Inc. (Northampton, MA) (16-20). The calorimeter cell (1.5 ml) was filled with reaction medium, and the reference cell was filled with MilliQ water. After equilibration at 35o C, the reaction was started by injecting vesicles into the reaction cell and the heat change during either Ca2+ uptake or Ca2+ efflux
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 one minute. The calorimetric enthalpy (∆Hcal) was calculated by dividing the amount of heat released by the amount of either substrate hydrolyzed or Ca2+ released by the vesicles. The units used were moles for substrate hydrolyzed and Ca2+ released and kcal for the heat released. A negative value indicates that the reaction is exothermic and a positive value indicates that it is endothermic.
Experimental procedure. All experiments were performed at 35oC. In a typical experiment the assay media 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, length of time and protein dilution as the vesicles kept in the calorimeter syringe. The different reactions were started
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 gluc. 6-P from ATP and glucose as previously described (20).
NaN3, an inhibitor of ATP synthase, and P1, P5-di(adenosine 5') pentaphosphate, a specific inhibitor of adenylate kinase, were added to the assay medium in order to avoid interference from possible contamination of the sarcoplasmic reticulum vesicles with these enzymes.
The free Ca2+ concentration in the medium was calculated using the association constants of Schwartzenbach et al. (27) in a computer program described by Fabiato & Fabiato (28) and modified by Sorenson et al. (29) ATP regenerating system. A large excess of pyruvate kinase, hexokinase or phosphofructokinase was used in order to assure that ATP was regenerated at a faster rate than it was cleaved by the Ca2+-ATPase. In control experiments the rates of substrate hydrolysis and Ca2+ uptake were measured in the presence of different concentrations of the ATP-regenerating enzymes and the concentration of enzyme used in all experiments described was 5 to 10 times higher than that needed for maximal activity (25, 26).