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Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase at submicromolar iron concentrations: evidence for metal-catalyzed oxidation
Free Radical Biology & Medicine, Vol. 25, Nos. 4/5, pp. 554 –560, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00084-7
Original Contribution OXIDATIVE DAMAGE TO SARCOPLASMIC RETICULUM Ca21-ATPase AT SUBMICROMOLAR IRON CONCENTRATIONS: EVIDENCE FOR METALCATALYZED OXIDATION VITOR HUGO MOREAU,* ROGER F. CASTILHO,† S´ERGIO T. FERREIRA,*
and
PAULO C. CARVALHO-ALVES*
*Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, and †Departamento de Patologia Clı´nica, Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas, Campinas, Brazil (Received 8 August 1997; Revised 19 February 1998; Accepted 30 March 1998)
Abstract—The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca21 pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe21 1 H2O2 3 HO• 1 HO2 1 Fe31). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca21-ATPase by oxygen reactive species at Fe21 concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe21, H2O2, and ascorbate. Measuring the rate constants of inactivation (kinact) at different Fe21 concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 mM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 mM Fe21. Inhibition was not affected by addition of 200 mM Ca21 to the medium, indicating that it was not related to iron binding to the high affinity Ca21 binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu21 was used instead of Fe21 in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca21-pump may be related to oxidative protein fragmentation mediated by site-specific Fe21 binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload. Keywords—Sarcoplasmic reticulum, ATPase, Oxidative damage, Oxidative stress, Iron overload, Fenton reaction, Free radical
INTRODUCTION
the permeability or uptake capacity of the reticulum. The sarcoplasmic reticulum (SR) is one of the best studied Ca21 transport systems from both structural and functional points of view,3– 6 being a very convenient model for investigation of the mechanism(s) of oxidative damage that lead to cytosolic Ca21 imbalance. Exposure of SR vesicles to Fe21/H2O2/ascorbate (a system that generates •OH radicals by Fenton chemistry: Fe21 1 H2O2 3 •OH 1 2OH 1 Fe31) caused inhibition of ATP hydrolysis and Ca21 uptake, which could not be prevented by blocking lipid peroxidation with BHT or by addition of the thiol reducing agent DTT.7 Inhibition was shown to be related to fragmentation of the ATPase polypeptide chain.7 Our present results indicate that inhibition of the SR ATPase involves Fe21 binding to a specific site in the enzyme, leading to polypeptide chain
Several cellular events are regulated by oscillations of cytosolic Ca21 concentration around 1027–1026 M. Abnormally higher intracellular Ca21 levels have been associated to mechanisms of cell injury promoted by chemical intoxicants and/or by reactive oxygen species generated under ischemia-reperfusion situations.1,2 Most of the Ca21 stored in the cell is accumulated in the endo(sarco)plasmic reticulum; thus, disturbances in cellular Ca21 homeostasis may result from modifications of Address correspondence to: P. C. Carvalho-Alves, Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil; Tel: (55-21) 270-5988; Fax: (55-21) 270-8647, E-Mail: pccalves@ bioqmed.ufrj.br. 554
Oxidative damage to SR calcium-ATPase
cleavage, without involving thiol oxidation. According to this mechanism, •OH radicals generated in situ rapidly react with neighboring aminoacid side-chains in the enzyme; thus, low Fe21 concentrations are required for extensive ATPase inhibition. This type of mechanism has been described as a metal-catalyzed oxidation.8 –10 These results also indicate a possible role of SR oxidative damage in the physiopathology of diseases associated with high iron levels in the organism (siderosis), as damage to endo(sarco)plasmic reticulum may occur at submicromolar or low micromolar Fe21 concentrations possibly reached under pathological conditions.
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SDS-PAGE Electrophoresis of SRV protein was performed in a discontinuous system according to Laemmli.15 The running gel was 7% in polyacrylamide and the stacking gel was 3%. SRV (100 mg protein/ml) were treated with oxidants for 30 min. Oxidation was arrested with 150 mM ophenantroline and membranes were solubilized with 2 volumes of loading buffer containing 250 mM Trisacetate, pH 7.4, 1.25 M sucrose, 5% SDS, and 10 mM EDTA. After boiling for 3 min, 0.1% bromophenol blue was added and aliquots containing 10 mg of protein were applied to each lane. After electrophoresis, the gel was silver stained.
MATERIALS AND METHODS
Enzyme preparation
Measurement of the rate of HO• production 11
SR vesicles were isolated from rabbit skeletal muscle. The ATPase content in the vesicles was approximately 80% based on total protein content, as indicated by SDS-PAGE stained with Coomasie Blue. Oxidative stress
SRV (50 mg/ml) were incubated in the presence of 0.5–10 mM Fe(NH3)2SO4, 20 –100 mM ascorbate, 20 mM HEPES-K1, pH 7.4, 80 mM KCl. Reaction was started at room temperature by addition of 0 –100 mM H2O2, in a final volume of 100 ml, and stopped by dilution of reaction medium in 700 ml of buffer containing 20 mM HEPES-K1, pH 7.4, 1 mM EDTA. Aliquots of 10 ml were withdrawn for determination of ATPase activity. ATPase activity This was assayed at 37°C by measuring 32Pi released after adsorption of nonhydrolyzed [g-32P]ATP on activated charcoal.12 The standard reaction medium (20 mM Hepes-K1, pH 7.4, 80 mM KCl, 4 mM MgCl2) contained 1 mM ATP and 50 mM free Ca21. Free Ca21 concentrations were calculated13 taking into account the pH and concentrations of K1, Mg21 and nucleotide, using a microcomputer program employing the dissociation constant for the Ca21-EGTA complex described by Schwartzenbach et al.14 To obtain the desired free Ca21 concentration, 0.32 mM CaCl2, 0.2 mM EGTA, and 0.08 mM EDTA were added to the medium. Reaction was started by addition of SR vesicles to a final protein concentration of 1 mg/ml and arrested after 60 min by addition of 3 volumes of a suspension of activated charcoal in 0.1 N HCl. After centrifugation, aliquots of the supernatant containing 32Pi were counted in a scintillation counter.
HO• production was measured by reaction with 2-deoxyD-ribose,16,17 with the following modifications: 50 mg/ml SRV were incubated for 0 –30 min in the presence of 1.5 mM Fe(NH3)2SO4, 100 mM ascorbate, 10 mM 2-deoxy-D-ribose, 20 mM HEPES-K1, pH 7.4. Reaction was started by addition of 100 mM H2O2 and stopped by addition of 1 volume of 4% o-phosphoric acid and 1 volume of 1% TBA. The sample was boiled for 10 min and formation of TBARS was followed by absorbance at 525 nm.18 Chemicals ATP, DTT, BHT, TBA, 2-deoxy-D-ribose, and ascorbate were from Sigma (St. Louis, MO). H2O2 was from Ecibra-Brazil. [32P]Pi was purchased from the Brazilian Institute for Nuclear Research (Sa˜o Paulo, SP, Brazil) and [g-32P]ATP was prepared as described.19 All other reagents were of the highest analytical grade available. RESULTS
In a previous study, we showed that the SR Ca21ATPase was inactivated when exposed to Fe21, H2O2, and ascorbate.7 However, the concentrations of oxidants used in that study were too high to be of physiopathological relevance. A more detailed investigation of the dependence on Fe21 concentration revealed that 80% inhibition of ATPase activity was observed when 50 mg/ml (;0.5 mM) ATPase was incubated for 7 min in the presence of 1 mM H2O2, 1 mM ascorbate, and micromolar Fe21 concentrations (Fig. 1). Control experiments in which SR ATPase was incubated in the presence of ascorbate alone (up to 5 mM) or ascorbate plus H2O2 (1 mM each, in the absence of iron) showed no inhibition of ATPase activity. Furthermore, incubation in
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Fig. 1. Fe21 dependence for ATPase inactivation. SRV (50 mg/ml) were incubated at room temperature in standard medium containing 20 mM HEPES-K1, pH 7.4, 80 mM KCl, 1 mM ascorbate, and the indicated Fe21 concentrations. Reaction was started by addition of 1 mM H2O2 and stopped after 7 min by 8-fold dilution in 20 mM HEPES-K1, pH 7.4, containing 1 mM EDTA. An aliquot was used to measure ATPase activity (see Materials and Methods). Symbols represent averages 6 standard deviations of three determinations.
the presence of up to 10 mM Fe21 alone also had no inhibitory effect. At Fe21 concentrations below 5 mM, longer times were required to completely inhibit the ATPase activity. After 30 min of incubation in the presence of 1.5 mM Fe21, maximal inhibition of ATPase activity was achieved with 20 mM ascorbate (Fig. 2). The time course of hydroxyl radical production (measured by degradation of deoxyribose) in the presence of 1.5 mM Fe21 was similar to the time course of ATPase inhibition (Fig. 3).
Fig. 2. Dependence on ascorbate concentration for ATPase inactivation. SRV were incubated in the presence of 1.5 mM Fe21 in standard medium containing the indicated ascorbate concentrations. Reaction was started, stopped and the ATPase activity measured as described in the legend to Fig. 1. The figure shows the result of a typical experiment.
Fig. 3. Time course of ATPase inactivation. SRV (50 mg/ml) were incubated with 1.5 mM Fe21, 100 mM ascorbate in standard medium. The reaction was started by addition of 20 mM (F) or 100 mM (E) H2O2, stopped at the indicated times, and ATPase activity was measured. The figure also shows the time course of •OH production in the presence of 100 mM H2O2 (ƒ) measured by deoxyribose degradation as described in Materials and Methods.
The deoxyribose degradation assay is an indirect method to evaluate •OH production by measuring the amount of TBARS released in the medium. TBARS could also be produced by lipid peroxidation of the SR membrane. However, we have carried out the deoxyribose assay both in the absence and presence of sarcoplasmic reticulum vesicles (SRV) and found that the amount of TBARS generated was not affected by presence of SRV. This indicates that, under our conditions, the extent of lipid peroxidation of the SR membrane was negligible relative to the amount of TBARS generated by deoxyribose degradation. Interestingly, similar time courses of ATPase inactivation were observed with either 20 or 100 mM H2O2 (Fig. 3), indicating that the kinetics of ATPase inactivation was of zero order with respect to H2O2 in this concentration range. If the concentration of Fe21 in the medium was varied in the presence of 100 mM H2O2 and 100 mM ascorbate, a family of double-exponential curves was obtained for the time courses of inactivation. The curves could be described by two exponential components, with a fast component (characteristic times of a few minutes) corresponding to 77–90% loss of activity. The second, minor inactivation component exhibited very slow kinetics (characteristic times of up to several hours) (Fig. 4A). Plotting the apparent inactivation constants (kinact) of the main exponential component as a function of Fe21 concentration resulted in a saturable curve from which an apparent K0.5 of ca. 2 mM was obtained (Fig. 4B), suggesting that inactivation could be due to Fe21 binding to a specific site in the ATPase, thus leading to localized oxidation. SDS-PAGE analysis of samples oxidized with
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Fig. 6. Effects of BHT, DTT, and DMSO on the ATPase inhibition induced by Fe21 or Cu21. SRV were incubated for 30 min in the presence of either 1.5 mM Fe21 or 1.5 mM Cu21, 100 mM H2O2 and 100 mM ascorbate. Where indicated, the medium also contained 30 mM BHT, 3 mM DTT, or 60 mM DMSO. ATPase activity was measured as described in Materials and Methods. Bars represent averages 6 standard deviations of four determinations.
Fig. 4. Time course of inactivation as a function of Fe21 concentration. (A) SRV were incubated in standard medium in the presence of 100 mM ascorbate and 0 (E), 0.5 (F), 1 (ƒ), 2 (), 5 (h), and 10 (h) mM Fe21. Reaction was started by addition of 100 mM H2O2, stopped at the indicated times, and ATPase activity was measured. Panel B shows the inactivation rate constant (see Results) as a function of Fe21 concentration.
the low Fe21 and H2O2 concentrations used in the present work showed a decrease in the amount of 100 kDa ATPase band (Fig. 5), confirming our previous
report of oxidative fragmentation of the ATPase polypeptide chain.7 To investigate whether oxidative inactivation could be related to Fe21 binding to the high-affinity Ca21 binding site of the ATPase, the effect of Ca21 on the inactivation was examined. Addition of 200 mM Ca21 had no effect on the time course or the extent of inactivation at 1.5 mM Fe21 (not shown), indicating that the two cations do not compete for the same binding site in the enzyme. Cu1 can replace Fe21 in the Fenton reaction and generates hydroxyl radicals more efficiently than Fe21 in aqueous solution.17 The ability of Cu1 to efficiently generate HO• radicals was checked under our experimental conditions by the deoxyribose degradation assay (not shown). In the presence of 1.5 mM Cu21, ATPase activity was even more inhibited than in the presence of an identical concentration of Fe21 (Fig. 6). With both iron and copper, no protection against inactivation was obtained with either BHT or the soluble hydroxyl radical scavenger DMSO (Fig. 6). However, in contrast to the inactivation produced by Fe21, inhibition by Cu1 probably involved oxidation of cysteine residues, as it could be completely prevented by DTT (Fig. 6). DISCUSSION
Fig. 5. SDS-PAGE analysis of oxidized ATPase. SRV (100 mg/ml) were exposed to 3 mM Fe21, 100 mM H2O2, and 300 mM ascorbate in standard medium for 30 min. Samples were then prepared for electrophoresis as described under Materials and Methods. Lane A shows control, non-oxidized SRV; Lane B shows oxidized SRV. The lower portion of the figure shows densitometric scans of control (dashed line) or oxidized (solid line) samples.
We have previously shown7 that impairment of SR ATPase function may occur under oxidative stress produced by Fe21/H2O2, a system that has been used to perform hydroxylation reactions.17 Because the ATPase is responsible for uptake of Ca21 into the reticulum, it rep-
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resents a good model for understanding pathological processes in which cytosolic Ca21 homeostasis is disrupted. Although several studies dealing with the SR ATPase and other Ca21-ATPases have demonstrated that oxidation of critical thiol groups in these enzymes caused loss of activity,20 –26 under the conditions employed in our study thiol oxidation did not seem responsible for ATPase inhibition by Fe21. This conclusion was based on the fact that addition of 3 mM DTT did not protect the ATPase against Fe21-induced oxidative inhibition. On the other hand, oxidative damage produced by incubation with 1.5 mM Cu21 instead of Fe21 could be totally prevented by DTT, indicating that the mechanism of inhibition by Cu1 likely involved thiol oxidation. Regarding a possible action of DTT as a reducing agent in regenerating Fe21 for the Fenton reaction, we note that we have worked under conditions of excess ascorbate (Fig. 2) included in the medium to reduce iron for the Fenton reaction. Furthermore, DTT was effective in protecting against copper-induced inactivation, but not against iron-induced inactivation, as stated above. If DTT acted predominantly as a reducing agent for the Fenton reaction one would expect that it would either potentiate or have no effect on ATPase inactivation by copper. Although our data indicate that different aminoacid residues are probably involved in the inactivation promoted by Fe21 or Cu21, the mechanism of oxidation may be similar with both metals, as water-soluble hydroxyl radical scavengers such as DMSO (Fig. 5) and mannitol (not shown) were not able to prevent oxidative damage to the enzyme by either Fe21 or Cu21. These results suggest that generation of reactive species that lead to ATPase inactivation may be spatially restricted to a metal binding site in the protein rather than in bulk solution, a type of mechanism that has been described as a metal-catalyzed protein oxidation.8 –10 Further evidence indicating that bound metal was involved in oxidative damage was obtained from the observation of a saturable dependence of the inactivation rate constant on Fe21 concentration (Fig. 4B) and from the finding that the rate of inactivation was of zero order with respect to H2O2 (Fig. 3). We have attempted to directly measure 55Fe binding to the ATPase using a filtration assay on 0.45 mm Millipore filters to retain the SR vesicles. However, using 55 Fe concentrations up to 50 mM, we were not able to detect significant binding to the ATPase under our experimental conditions (data not shown). Considering the detection limits of the methodology we have used, these results indicate that the occupancy of ATPase binding sites by iron was less than 0.1 mol of bound iron/mol of ATPase. These results may be explained by assuming
that in situ generation of hydroxyl radicals by Fenton chemistry is so efficient that it would not require a long residence time of iron at its binding site in the ATPase molecule, resulting in low occupancy of binding sites. This is in line with results obtained from comparison of the rates of deoxyribose degradation and ATPase inactivation (Fig. 3, see below), and indicates that in equilibrium most of the iron is free in solution rather than bound to the ATPase. This could also mean that a high-affinity binding site is not an absolute requirement for metalcatalyzed protein oxidation. Instead, proper coordination of iron by neighboring aminoacids at the binding site may be essential to allow fast, efficient oxidation. The time course for ATPase inactivation in the presence of Fe21 was similar to the time course of deoxyribose degradation under the same experimental conditions (Fig. 3). This was a surprising result since •OH radicals detected by the deoxyribose assay are those produced in the bulk of the solution, and not necessarily the ones that mediate damage. In fact, the velocity of ATPase inactivation is given by v1 5 k1[ATPase][•OH], and the velocity of deoxyribose degradation is given by v2 5 k2[deoxyribose][•OH], where k1 and k2 are the rate constants for •OH reaction with ATPase or deoxyribose, respectively. Because in the conditions employed in the experiment shown in Fig. 3 the concentration of deoxyribose was 2000-fold higher than the concentration of ATPase, the similarity in the time courses suggests that the efficiency of ATPase inactivation was much higher than the efficiency of deoxyribose degradation. This could indicate that k1 and/or the effective concentration of •OH for ATPase inactivation were much higher than would be expected if •OH radicals were generated freely in bulk solution and reacted with ATPase by diffusion. These observations are consistent with generation of •OH at a metal binding site in the ATPase, which would allow for fast, efficient attack of nearby aminoacid side-chains, in an environment protected from water-soluble scavengers. Oxidative damage could then lead to protein inactivation and fragmentation. Protein fragmentation induced by oxygen reactive species has been described.27–31 In the case of metal-ion catalyzed oxidation, it has been proposed that oxidation of proline residues to 2-pyrrolidone32 may provide a mechanism for peptide bond cleavage.10 The site of oxidation could be the catalytic ATPase site rather than the calcium transport sites, as addition of excess Ca21 to the medium did not prevent inhibition. This is in line with our previous finding7 that concentrations of trifluoperazine that block phosphorylation of the catalytic site of the ATPase33 confer complete protection from oxidation. More recently, ATP protection of cardiac sarcoplasmic reticulum ATPase from inactivation by a very similar oxidation system has been described.34
Oxidative damage to SR calcium-ATPase
Possible pathological implications Local or generalized iron overload may occur in humans in many different situations in which clinical tissue injury is present.35 Although most of the intracellular iron is stored bound to high molecular weight proteins (ferritins and hemosiderins), iron binding to small molecules (nucleotides, metabolic intermediates, or polypeptides) may also occur. This low molecular weight iron pool may reach 0.5 to 1.0 mM,36 and its role in generating oxidative damage associated to several pathological processes has been extensively proposed.36,37 Thus, it seems possible that excess iron could bind to other cell components, leading, among other consequences, to oxidative inactivation of the Ca21-ATPase (which, as shown here, requires very low iron concentrations), disruption of Ca21 homeostasis, and ultimately to cell death.1,2,38 Acknowledgements—This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro, and Programa de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico to S. T. F. V. H. M. is supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal Docente de Ensino Superior. S. T. F. is a Howard Hughes Medical Institute International Research Scholar.
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ABBREVIATIONS
BHT— butylated hydroxytoluene DMSO— dimethylsulfoxide
DTT— dithiothreitol EDTA— ethylenediaminetetraacetic acid Hepes— 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid SDS—sodium dodecylsulfate SDS-PAGE—polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate SR—sarcoplasmic reticulum SRV—sarcoplasmic reticulum vesicles TBA—thiobarbituric acid TBARS—thiobarbituric acid-reactive substances
PII S0891-5849(98)00084-7
Original Contribution OXIDATIVE DAMAGE TO SARCOPLASMIC RETICULUM Ca21-ATPase AT SUBMICROMOLAR IRON CONCENTRATIONS: EVIDENCE FOR METALCATALYZED OXIDATION VITOR HUGO MOREAU,* ROGER F. CASTILHO,† S´ERGIO T. FERREIRA,*
and
PAULO C. CARVALHO-ALVES*
*Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, and †Departamento de Patologia Clı´nica, Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas, Campinas, Brazil (Received 8 August 1997; Revised 19 February 1998; Accepted 30 March 1998)
Abstract—The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca21 pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe21 1 H2O2 3 HO• 1 HO2 1 Fe31). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca21-ATPase by oxygen reactive species at Fe21 concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe21, H2O2, and ascorbate. Measuring the rate constants of inactivation (kinact) at different Fe21 concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 mM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 mM Fe21. Inhibition was not affected by addition of 200 mM Ca21 to the medium, indicating that it was not related to iron binding to the high affinity Ca21 binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu21 was used instead of Fe21 in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca21-pump may be related to oxidative protein fragmentation mediated by site-specific Fe21 binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload. Keywords—Sarcoplasmic reticulum, ATPase, Oxidative damage, Oxidative stress, Iron overload, Fenton reaction, Free radical
INTRODUCTION
the permeability or uptake capacity of the reticulum. The sarcoplasmic reticulum (SR) is one of the best studied Ca21 transport systems from both structural and functional points of view,3– 6 being a very convenient model for investigation of the mechanism(s) of oxidative damage that lead to cytosolic Ca21 imbalance. Exposure of SR vesicles to Fe21/H2O2/ascorbate (a system that generates •OH radicals by Fenton chemistry: Fe21 1 H2O2 3 •OH 1 2OH 1 Fe31) caused inhibition of ATP hydrolysis and Ca21 uptake, which could not be prevented by blocking lipid peroxidation with BHT or by addition of the thiol reducing agent DTT.7 Inhibition was shown to be related to fragmentation of the ATPase polypeptide chain.7 Our present results indicate that inhibition of the SR ATPase involves Fe21 binding to a specific site in the enzyme, leading to polypeptide chain
Several cellular events are regulated by oscillations of cytosolic Ca21 concentration around 1027–1026 M. Abnormally higher intracellular Ca21 levels have been associated to mechanisms of cell injury promoted by chemical intoxicants and/or by reactive oxygen species generated under ischemia-reperfusion situations.1,2 Most of the Ca21 stored in the cell is accumulated in the endo(sarco)plasmic reticulum; thus, disturbances in cellular Ca21 homeostasis may result from modifications of Address correspondence to: P. C. Carvalho-Alves, Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil; Tel: (55-21) 270-5988; Fax: (55-21) 270-8647, E-Mail: pccalves@ bioqmed.ufrj.br. 554
Oxidative damage to SR calcium-ATPase
cleavage, without involving thiol oxidation. According to this mechanism, •OH radicals generated in situ rapidly react with neighboring aminoacid side-chains in the enzyme; thus, low Fe21 concentrations are required for extensive ATPase inhibition. This type of mechanism has been described as a metal-catalyzed oxidation.8 –10 These results also indicate a possible role of SR oxidative damage in the physiopathology of diseases associated with high iron levels in the organism (siderosis), as damage to endo(sarco)plasmic reticulum may occur at submicromolar or low micromolar Fe21 concentrations possibly reached under pathological conditions.
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SDS-PAGE Electrophoresis of SRV protein was performed in a discontinuous system according to Laemmli.15 The running gel was 7% in polyacrylamide and the stacking gel was 3%. SRV (100 mg protein/ml) were treated with oxidants for 30 min. Oxidation was arrested with 150 mM ophenantroline and membranes were solubilized with 2 volumes of loading buffer containing 250 mM Trisacetate, pH 7.4, 1.25 M sucrose, 5% SDS, and 10 mM EDTA. After boiling for 3 min, 0.1% bromophenol blue was added and aliquots containing 10 mg of protein were applied to each lane. After electrophoresis, the gel was silver stained.
MATERIALS AND METHODS
Enzyme preparation
Measurement of the rate of HO• production 11
SR vesicles were isolated from rabbit skeletal muscle. The ATPase content in the vesicles was approximately 80% based on total protein content, as indicated by SDS-PAGE stained with Coomasie Blue. Oxidative stress
SRV (50 mg/ml) were incubated in the presence of 0.5–10 mM Fe(NH3)2SO4, 20 –100 mM ascorbate, 20 mM HEPES-K1, pH 7.4, 80 mM KCl. Reaction was started at room temperature by addition of 0 –100 mM H2O2, in a final volume of 100 ml, and stopped by dilution of reaction medium in 700 ml of buffer containing 20 mM HEPES-K1, pH 7.4, 1 mM EDTA. Aliquots of 10 ml were withdrawn for determination of ATPase activity. ATPase activity This was assayed at 37°C by measuring 32Pi released after adsorption of nonhydrolyzed [g-32P]ATP on activated charcoal.12 The standard reaction medium (20 mM Hepes-K1, pH 7.4, 80 mM KCl, 4 mM MgCl2) contained 1 mM ATP and 50 mM free Ca21. Free Ca21 concentrations were calculated13 taking into account the pH and concentrations of K1, Mg21 and nucleotide, using a microcomputer program employing the dissociation constant for the Ca21-EGTA complex described by Schwartzenbach et al.14 To obtain the desired free Ca21 concentration, 0.32 mM CaCl2, 0.2 mM EGTA, and 0.08 mM EDTA were added to the medium. Reaction was started by addition of SR vesicles to a final protein concentration of 1 mg/ml and arrested after 60 min by addition of 3 volumes of a suspension of activated charcoal in 0.1 N HCl. After centrifugation, aliquots of the supernatant containing 32Pi were counted in a scintillation counter.
HO• production was measured by reaction with 2-deoxyD-ribose,16,17 with the following modifications: 50 mg/ml SRV were incubated for 0 –30 min in the presence of 1.5 mM Fe(NH3)2SO4, 100 mM ascorbate, 10 mM 2-deoxy-D-ribose, 20 mM HEPES-K1, pH 7.4. Reaction was started by addition of 100 mM H2O2 and stopped by addition of 1 volume of 4% o-phosphoric acid and 1 volume of 1% TBA. The sample was boiled for 10 min and formation of TBARS was followed by absorbance at 525 nm.18 Chemicals ATP, DTT, BHT, TBA, 2-deoxy-D-ribose, and ascorbate were from Sigma (St. Louis, MO). H2O2 was from Ecibra-Brazil. [32P]Pi was purchased from the Brazilian Institute for Nuclear Research (Sa˜o Paulo, SP, Brazil) and [g-32P]ATP was prepared as described.19 All other reagents were of the highest analytical grade available. RESULTS
In a previous study, we showed that the SR Ca21ATPase was inactivated when exposed to Fe21, H2O2, and ascorbate.7 However, the concentrations of oxidants used in that study were too high to be of physiopathological relevance. A more detailed investigation of the dependence on Fe21 concentration revealed that 80% inhibition of ATPase activity was observed when 50 mg/ml (;0.5 mM) ATPase was incubated for 7 min in the presence of 1 mM H2O2, 1 mM ascorbate, and micromolar Fe21 concentrations (Fig. 1). Control experiments in which SR ATPase was incubated in the presence of ascorbate alone (up to 5 mM) or ascorbate plus H2O2 (1 mM each, in the absence of iron) showed no inhibition of ATPase activity. Furthermore, incubation in
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Fig. 1. Fe21 dependence for ATPase inactivation. SRV (50 mg/ml) were incubated at room temperature in standard medium containing 20 mM HEPES-K1, pH 7.4, 80 mM KCl, 1 mM ascorbate, and the indicated Fe21 concentrations. Reaction was started by addition of 1 mM H2O2 and stopped after 7 min by 8-fold dilution in 20 mM HEPES-K1, pH 7.4, containing 1 mM EDTA. An aliquot was used to measure ATPase activity (see Materials and Methods). Symbols represent averages 6 standard deviations of three determinations.
the presence of up to 10 mM Fe21 alone also had no inhibitory effect. At Fe21 concentrations below 5 mM, longer times were required to completely inhibit the ATPase activity. After 30 min of incubation in the presence of 1.5 mM Fe21, maximal inhibition of ATPase activity was achieved with 20 mM ascorbate (Fig. 2). The time course of hydroxyl radical production (measured by degradation of deoxyribose) in the presence of 1.5 mM Fe21 was similar to the time course of ATPase inhibition (Fig. 3).
Fig. 2. Dependence on ascorbate concentration for ATPase inactivation. SRV were incubated in the presence of 1.5 mM Fe21 in standard medium containing the indicated ascorbate concentrations. Reaction was started, stopped and the ATPase activity measured as described in the legend to Fig. 1. The figure shows the result of a typical experiment.
Fig. 3. Time course of ATPase inactivation. SRV (50 mg/ml) were incubated with 1.5 mM Fe21, 100 mM ascorbate in standard medium. The reaction was started by addition of 20 mM (F) or 100 mM (E) H2O2, stopped at the indicated times, and ATPase activity was measured. The figure also shows the time course of •OH production in the presence of 100 mM H2O2 (ƒ) measured by deoxyribose degradation as described in Materials and Methods.
The deoxyribose degradation assay is an indirect method to evaluate •OH production by measuring the amount of TBARS released in the medium. TBARS could also be produced by lipid peroxidation of the SR membrane. However, we have carried out the deoxyribose assay both in the absence and presence of sarcoplasmic reticulum vesicles (SRV) and found that the amount of TBARS generated was not affected by presence of SRV. This indicates that, under our conditions, the extent of lipid peroxidation of the SR membrane was negligible relative to the amount of TBARS generated by deoxyribose degradation. Interestingly, similar time courses of ATPase inactivation were observed with either 20 or 100 mM H2O2 (Fig. 3), indicating that the kinetics of ATPase inactivation was of zero order with respect to H2O2 in this concentration range. If the concentration of Fe21 in the medium was varied in the presence of 100 mM H2O2 and 100 mM ascorbate, a family of double-exponential curves was obtained for the time courses of inactivation. The curves could be described by two exponential components, with a fast component (characteristic times of a few minutes) corresponding to 77–90% loss of activity. The second, minor inactivation component exhibited very slow kinetics (characteristic times of up to several hours) (Fig. 4A). Plotting the apparent inactivation constants (kinact) of the main exponential component as a function of Fe21 concentration resulted in a saturable curve from which an apparent K0.5 of ca. 2 mM was obtained (Fig. 4B), suggesting that inactivation could be due to Fe21 binding to a specific site in the ATPase, thus leading to localized oxidation. SDS-PAGE analysis of samples oxidized with
Oxidative damage to SR calcium-ATPase
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Fig. 6. Effects of BHT, DTT, and DMSO on the ATPase inhibition induced by Fe21 or Cu21. SRV were incubated for 30 min in the presence of either 1.5 mM Fe21 or 1.5 mM Cu21, 100 mM H2O2 and 100 mM ascorbate. Where indicated, the medium also contained 30 mM BHT, 3 mM DTT, or 60 mM DMSO. ATPase activity was measured as described in Materials and Methods. Bars represent averages 6 standard deviations of four determinations.
Fig. 4. Time course of inactivation as a function of Fe21 concentration. (A) SRV were incubated in standard medium in the presence of 100 mM ascorbate and 0 (E), 0.5 (F), 1 (ƒ), 2 (), 5 (h), and 10 (h) mM Fe21. Reaction was started by addition of 100 mM H2O2, stopped at the indicated times, and ATPase activity was measured. Panel B shows the inactivation rate constant (see Results) as a function of Fe21 concentration.
the low Fe21 and H2O2 concentrations used in the present work showed a decrease in the amount of 100 kDa ATPase band (Fig. 5), confirming our previous
report of oxidative fragmentation of the ATPase polypeptide chain.7 To investigate whether oxidative inactivation could be related to Fe21 binding to the high-affinity Ca21 binding site of the ATPase, the effect of Ca21 on the inactivation was examined. Addition of 200 mM Ca21 had no effect on the time course or the extent of inactivation at 1.5 mM Fe21 (not shown), indicating that the two cations do not compete for the same binding site in the enzyme. Cu1 can replace Fe21 in the Fenton reaction and generates hydroxyl radicals more efficiently than Fe21 in aqueous solution.17 The ability of Cu1 to efficiently generate HO• radicals was checked under our experimental conditions by the deoxyribose degradation assay (not shown). In the presence of 1.5 mM Cu21, ATPase activity was even more inhibited than in the presence of an identical concentration of Fe21 (Fig. 6). With both iron and copper, no protection against inactivation was obtained with either BHT or the soluble hydroxyl radical scavenger DMSO (Fig. 6). However, in contrast to the inactivation produced by Fe21, inhibition by Cu1 probably involved oxidation of cysteine residues, as it could be completely prevented by DTT (Fig. 6). DISCUSSION
Fig. 5. SDS-PAGE analysis of oxidized ATPase. SRV (100 mg/ml) were exposed to 3 mM Fe21, 100 mM H2O2, and 300 mM ascorbate in standard medium for 30 min. Samples were then prepared for electrophoresis as described under Materials and Methods. Lane A shows control, non-oxidized SRV; Lane B shows oxidized SRV. The lower portion of the figure shows densitometric scans of control (dashed line) or oxidized (solid line) samples.
We have previously shown7 that impairment of SR ATPase function may occur under oxidative stress produced by Fe21/H2O2, a system that has been used to perform hydroxylation reactions.17 Because the ATPase is responsible for uptake of Ca21 into the reticulum, it rep-
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resents a good model for understanding pathological processes in which cytosolic Ca21 homeostasis is disrupted. Although several studies dealing with the SR ATPase and other Ca21-ATPases have demonstrated that oxidation of critical thiol groups in these enzymes caused loss of activity,20 –26 under the conditions employed in our study thiol oxidation did not seem responsible for ATPase inhibition by Fe21. This conclusion was based on the fact that addition of 3 mM DTT did not protect the ATPase against Fe21-induced oxidative inhibition. On the other hand, oxidative damage produced by incubation with 1.5 mM Cu21 instead of Fe21 could be totally prevented by DTT, indicating that the mechanism of inhibition by Cu1 likely involved thiol oxidation. Regarding a possible action of DTT as a reducing agent in regenerating Fe21 for the Fenton reaction, we note that we have worked under conditions of excess ascorbate (Fig. 2) included in the medium to reduce iron for the Fenton reaction. Furthermore, DTT was effective in protecting against copper-induced inactivation, but not against iron-induced inactivation, as stated above. If DTT acted predominantly as a reducing agent for the Fenton reaction one would expect that it would either potentiate or have no effect on ATPase inactivation by copper. Although our data indicate that different aminoacid residues are probably involved in the inactivation promoted by Fe21 or Cu21, the mechanism of oxidation may be similar with both metals, as water-soluble hydroxyl radical scavengers such as DMSO (Fig. 5) and mannitol (not shown) were not able to prevent oxidative damage to the enzyme by either Fe21 or Cu21. These results suggest that generation of reactive species that lead to ATPase inactivation may be spatially restricted to a metal binding site in the protein rather than in bulk solution, a type of mechanism that has been described as a metal-catalyzed protein oxidation.8 –10 Further evidence indicating that bound metal was involved in oxidative damage was obtained from the observation of a saturable dependence of the inactivation rate constant on Fe21 concentration (Fig. 4B) and from the finding that the rate of inactivation was of zero order with respect to H2O2 (Fig. 3). We have attempted to directly measure 55Fe binding to the ATPase using a filtration assay on 0.45 mm Millipore filters to retain the SR vesicles. However, using 55 Fe concentrations up to 50 mM, we were not able to detect significant binding to the ATPase under our experimental conditions (data not shown). Considering the detection limits of the methodology we have used, these results indicate that the occupancy of ATPase binding sites by iron was less than 0.1 mol of bound iron/mol of ATPase. These results may be explained by assuming
that in situ generation of hydroxyl radicals by Fenton chemistry is so efficient that it would not require a long residence time of iron at its binding site in the ATPase molecule, resulting in low occupancy of binding sites. This is in line with results obtained from comparison of the rates of deoxyribose degradation and ATPase inactivation (Fig. 3, see below), and indicates that in equilibrium most of the iron is free in solution rather than bound to the ATPase. This could also mean that a high-affinity binding site is not an absolute requirement for metalcatalyzed protein oxidation. Instead, proper coordination of iron by neighboring aminoacids at the binding site may be essential to allow fast, efficient oxidation. The time course for ATPase inactivation in the presence of Fe21 was similar to the time course of deoxyribose degradation under the same experimental conditions (Fig. 3). This was a surprising result since •OH radicals detected by the deoxyribose assay are those produced in the bulk of the solution, and not necessarily the ones that mediate damage. In fact, the velocity of ATPase inactivation is given by v1 5 k1[ATPase][•OH], and the velocity of deoxyribose degradation is given by v2 5 k2[deoxyribose][•OH], where k1 and k2 are the rate constants for •OH reaction with ATPase or deoxyribose, respectively. Because in the conditions employed in the experiment shown in Fig. 3 the concentration of deoxyribose was 2000-fold higher than the concentration of ATPase, the similarity in the time courses suggests that the efficiency of ATPase inactivation was much higher than the efficiency of deoxyribose degradation. This could indicate that k1 and/or the effective concentration of •OH for ATPase inactivation were much higher than would be expected if •OH radicals were generated freely in bulk solution and reacted with ATPase by diffusion. These observations are consistent with generation of •OH at a metal binding site in the ATPase, which would allow for fast, efficient attack of nearby aminoacid side-chains, in an environment protected from water-soluble scavengers. Oxidative damage could then lead to protein inactivation and fragmentation. Protein fragmentation induced by oxygen reactive species has been described.27–31 In the case of metal-ion catalyzed oxidation, it has been proposed that oxidation of proline residues to 2-pyrrolidone32 may provide a mechanism for peptide bond cleavage.10 The site of oxidation could be the catalytic ATPase site rather than the calcium transport sites, as addition of excess Ca21 to the medium did not prevent inhibition. This is in line with our previous finding7 that concentrations of trifluoperazine that block phosphorylation of the catalytic site of the ATPase33 confer complete protection from oxidation. More recently, ATP protection of cardiac sarcoplasmic reticulum ATPase from inactivation by a very similar oxidation system has been described.34
Oxidative damage to SR calcium-ATPase
Possible pathological implications Local or generalized iron overload may occur in humans in many different situations in which clinical tissue injury is present.35 Although most of the intracellular iron is stored bound to high molecular weight proteins (ferritins and hemosiderins), iron binding to small molecules (nucleotides, metabolic intermediates, or polypeptides) may also occur. This low molecular weight iron pool may reach 0.5 to 1.0 mM,36 and its role in generating oxidative damage associated to several pathological processes has been extensively proposed.36,37 Thus, it seems possible that excess iron could bind to other cell components, leading, among other consequences, to oxidative inactivation of the Ca21-ATPase (which, as shown here, requires very low iron concentrations), disruption of Ca21 homeostasis, and ultimately to cell death.1,2,38 Acknowledgements—This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro, and Programa de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico to S. T. F. V. H. M. is supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal Docente de Ensino Superior. S. T. F. is a Howard Hughes Medical Institute International Research Scholar.
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ABBREVIATIONS
BHT— butylated hydroxytoluene DMSO— dimethylsulfoxide
DTT— dithiothreitol EDTA— ethylenediaminetetraacetic acid Hepes— 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid SDS—sodium dodecylsulfate SDS-PAGE—polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate SR—sarcoplasmic reticulum SRV—sarcoplasmic reticulum vesicles TBA—thiobarbituric acid TBARS—thiobarbituric acid-reactive substances