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(Circulation Research. 1997;80:76-81.)
© 1997 American Heart Association, Inc.

Articles

Hydroxyl Radical Inhibits Sarcoplasmic Reticulum Ca²⁺-ATPase Function by Direct Attack on the ATP Binding Site

Kai Y. Xu, Jay L. Zweier, Lewis C. Becker

the Department of Medicine, Division of Cardiology, The Johns Hopkins Medical Institutions, Baltimore, Md.

Correspondence to Dr Kai Y. Xu, The Johns Hopkins Medical Institutions, Department of Medicine, Cardiology Division, 5501 Hopkins Bayview Circle, Room 3A-29, Baltimore, MD 21224.

	Abstract

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Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca²⁺-ATPase, potentially contributing to cellular Ca²⁺ overload and myocardial damage after ischemia and reperfusion. To determine whether the ATP binding site on Ca²⁺-ATPase is involved in oxygen radical injury, SR vesicles containing bound Ca²⁺-ATPase were isolated from rabbit cardiac and skeletal muscle and exposed to a hydroxyl radical (·OH)–generating system consisting of H₂O₂ and Fe³⁺-nitrilotriacetic acid in amounts that generate a magnitude of ·OH similar to that which occurs in the reperfused heart. ·OH exposure completely inhibited Ca²⁺-ATPase activity and SR ⁴⁵Ca uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with l mmol/L ATP before exposure to ·OH, complete protection was observed: there was no loss of ATPase activity or ⁴⁵Ca transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (l mmol/L each). SDS–gel electrophoresis indicated that ·OH did not damage the primary structure of the enzyme. Electron paramagnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge ·OH. These results suggest that ·OH denatures the SR Ca²⁺-ATPase by directly attacking the ATP binding site, and occupation of the active site by ATP protects against ·OH-induced loss of enzymatic activity and SR Ca²⁺ transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of ·OH at the time of reperfusion.

Key Words: sarcoplasmic reticulum • Ca²⁺-ATPase • ATP • hydroxyl radical • ischemia/reperfusion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Highly reactive oxidants and oxygen-derived free radicals, including the superoxide anion radical, hydrogen peroxide, hydroxyl radical, and singlet oxygen, are generated after myocardial ischemia/reperfusion and have been implicated as important factors in the pathogenesis of cellular injury in the postischemic heart.^{1 2 3} The sequestration of cytoplasmic Ca²⁺ into the SR is mediated by a Mg²⁺-dependent Ca²⁺-ATPase; a decrease in SR Ca²⁺-ATPase function appears to contribute to the intracellular Ca²⁺ overload that accompanies myocardial damage after myocardial ischemia and reperfusion.^{4 5} A reduction in SR Ca²⁺-ATPase activity has been shown to occur after ischemia/reperfusion.^{6 7} It has also been reported that a decrease in Ca²⁺ uptake occurs in isolated SR after exposure to xanthine/xanthine oxidase, an enzymatic system capable of generating superoxide radicals,⁸ or to activated neutrophils, which may release large amounts of superoxide, hypochlorous acid, and other oxidants. However, the precise molecular mechanism involved in SR Ca²⁺-ATPase inhibition by oxygen radicals and the site of free radical attack are not known.

The present study was undertaken to investigate whether hydroxyl free radical (·OH)–induced SR dysfunction occurs as a result of a direct attack on the ATP binding site of the Ca²⁺-ATPase. The hydroxyl free radical was chosen for our experiments because it is generated in the postischemic heart and has been shown to result in contractile dysfunction.^{9 10} With exogenous administration of ·OH to isolated hearts or myocytes, SR function and Ca²⁺ homeostasis are affected, and a cellular Ca²⁺-overload state occurs that is similar to that seen after ischemia and reperfusion.¹¹ In the present study, we demonstrate that ·OH generation similar in magnitude to that measured during the early minutes of postischemic reperfusion denatures the Ca²⁺-ATPase. We show that presaturation of the active site with ATP completely protects both cardiac and skeletal muscle SR Ca²⁺-ATPase function from hydroxyl radical–induced inhibition. This observation suggests that ·OH free radicals inhibit SR Ca²⁺-ATPase by directly damaging the ATP active site rather than producing a generalized denaturation of the enzyme.

	Materials and Methods

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All reagents were purchased from Sigma Chemical Co, unless specified. ⁴⁵Ca was from DuPont Research Products. The spin trap DMPO was purchased from Aldrich Chemical Co and further purified by double distillation, as reported previously.¹²

Purification of SR Vesicles Containing Bound Ca²⁺-ATPase
Cardiac and skeletal muscle SR vesicles were prepared from hearts and hind legs of New Zealand White rabbits according to the method of Chu et al,¹³ with modifications similar to those described previously.¹⁴ Briefly, the initial homogenization was carried out twice in 0.29 mol/L sucrose, 0.6 mol/L KCl, and 10 mmol/L imidazole/HCl buffer, pH 7.2, for 15 seconds at 22 000 rpm using an OMNI 5000 tissue homogenizer. The homogenate was centrifuged at 8000 rpm for 15 minutes, and the pellet was discarded. The supernatant was then centrifuged at 30 000 rpm for 90 minutes, and the pellet was harvested, resuspended, and loaded on top of a sucrose step gradient (45%, 38%, 34%, 32%, 26%, and 20%). Centrifugation was carried out for 16 hours at 20 000 rpm with an SW-28 Beckman rotor. The SR fraction at the interfaces between 32% and 34% gradient steps was collected and sedimented for 90 minutes at 32 000 rpm. The final pellet was resuspended in 10 mmol/L imidazole/HCl and 0.29 mol/L sucrose buffer, pH 7.2, and stored at -70°C. Protein concentration was determined by the method of Lowry et al.¹⁵

Hydroxyl Radical–Generating System
Hydroxyl radical (·OH) was generated from a system consisting of H₂O₂ and the ferric iron chelate Fe³⁺-NTA. The Fe³⁺-NTA was prepared as described previously.¹⁶ A final concentration of 1 mmol/L H₂O₂ was used in all experiments. We have previously demonstrated that the ·OH-generating system consisting of 1 mmol/L H₂O₂ and 100 µmol/L Fe³⁺-NTA when infused into isolated rabbit hearts generates a magnitude of ·OH similar to that observed during the early minutes of postischemic reperfusion.¹⁷ Spin-trapping measurements of ·OH formation from infusion of this generating system in the isolated rabbit heart gave rise to magnitudes of DMPO-OH adduct signals similar to those observed during the early period of postischemic reflow, with DMPO-OH adduct concentrations of 1 µmol/L observed. The Fenton chemistry¹⁸ by which ·OH is generated proceeds according to following reactions:

Determination of Ca²⁺-ATPase Activity
The ATPase assay procedure was a modification of the method of Kyte.¹⁹ The enzymatic activity was defined as the Tg-sensitive hydrolysis of Mg²⁺-ATP¹³ in the presence of Ca²⁺ (10 µmol/L) for both cardiac and skeletal SR Ca²⁺-ATPase. Briefly, the incubation mixture contained 15 mmol/L imidazole/HCl, pH 7.4, 1 mmol/L ATP, and 10 mmol/L Mg²⁺ in a final volume of 0.5 mL and was brought to and maintained at 37°C in a water bath. The reaction was initiated by adding SR vesicles at 37°C and stopped after 25 minutes by adding 0.75 mL quench solution (0.5% ammonium molybdate+0.5 mol/L H₂SO₄) and 0.02 mL developer (25 mg/mL of a mixture of 0.2 g 1-amino-2-naphthol-4-sulfonic acid+1.2 g sodium bisulfate+1.2 g sodium sulfite). The color was allowed to develop for 30 minutes at room temperature, and the phosphate generated in the reaction was then determined at 700 nm using a spectrophotometer. For experiments involving exposure to ·OH, SR vesicles were premixed either with ADP, AMP, adenosine, or sucrose (1 mmol/L each) for 3 minutes, and various concentrations of the ·OH-generating system were then added for 15 minutes at room temperature before adding ATP to start the reaction, which was quenched as above after 25 minutes. For the ATP protection experiment, ATP (1 mmol/L) was preincubated with the vesicles for 3 minutes at room temperature, and various concentrations of the ·OH-generating system were then added to the sample mixture (1 mmol/L H₂O₂+20 to 200 µmol/L Fe³⁺-NTA as indicated in Fig 1) and quenched after 25 minutes. For dose-dependent ATP protection experiments, purified cardiac SR (10 µg/mL) was incubated with various concentrations of ATP (10 nmol/L to 10 mmol/L) in the presence or absence of the ·OH-generating system (0.2 mmol/L Fe³⁺-NTA+1 mmol/L H₂O₂) for 30 minutes at room temperature; an additional 2 mmol/L ATP was added to each sample, and the ATPase assay was then carried out at 37°C for 30 minutes. All of the experiments were performed in the presence and absence of Tg, a specific inhibitor of SR Ca²⁺-ATPase. Any ATPase activity remaining in the presence of Tg was considered to represent nonspecific activity. The specific activity of the SR Ca²⁺-ATPase used in these studies was 200 µmol Pi/mg per hour for cardiac Ca²⁺-ATPase and 600 µmol Pi/mg per hour for skeletal muscle Ca²⁺-ATPase.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of AMP, ADP, ATP, adenosine (Ad), and sucrose (Su) on the inactivation of cardiac and skeletal muscle SR Ca2+-ATPase by hydroxyl free radical. Purified cardiac (35 µg/mL) and skeletal (14 µg/mL) muscle SR were premixed with the hydroxyl free radical–generating system (H2O2+Fe3+-NTA) in the presence or absence of one of the above ligands, as described in "Materials and Methods." The symbols represent each individual experiment for both cardiac and skeletal muscle SR Ca2+-ATPase as indicated. Data represent the mean of three experiments. ATPase activity was completely protected by 1 mmol/L ATP for both cardiac and skeletal muscle SR Ca2+-ATPase but not by ADP, AMP, Ad, or Su (1 mmol/L each).

⁴⁵Ca Uptake by SR Vesicles
In addition to cardiac (0.28 mg/mL) or skeletal muscle (0.29 mg/mL) SR, the reaction mixture contained Ca²⁺ (10 µmol/L), ⁴⁵Ca (1 µCi/mL), Mg²⁺ (8 mmol/L), AMP, ADP, and ATP (1 mmol/L each). The ·OH-generating system (1 mmol/L H₂O₂ and 0.2 mmol/L Fe³⁺-NTA) was added, and samples were incubated in the presence and absence of Tg at 37°C for 15 minutes. The reaction was stopped by adding Tg (20 µmol/L) and pelleting the sample at 14 000 rpm for 15 minutes. The pellet was washed three times with 15 mmol/L imidazole/HCl buffer, pH 7.2, and then dissolved in 100 µL of 10% SDS solution. The radioactivity was determined by a ß-scintillation counter. The amount of inactivation of Ca²⁺ uptake caused by ·OH was calculated by comparison with uptake in the absence of ·OH under the same experimental conditions. Any ⁴⁵Ca transport in the presence of Tg was considered to represent nonspecific ⁴⁵Ca uptake.

EPR Measurements of Free Radical Generation
EPR spin-trapping measurements were performed in the presence of the spin trap DMPO²⁰ and recorded at room temperature for 30 minutes using an IBM-Bruker ER 300 spectrometer operating at X-band with a TM₁₁₀ cavity and flat cell. The spectrometer settings were as follows: modulation frequency, 100 kHz; modulation amplitude, 1 G; scan time, 1.0 minutes; microwave power, 20 mW; and microwave frequency, 100 kHz. Repetitive 1-minute acquisitions were performed, and the digitized Bruker spectral data were transferred to personal computer for analysis. Spectral simulations were performed on the personal computer and directly matched with the experimental data to extract the spectral parameters, as described previously.¹¹

	Results

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Inactivation of SR Ca²⁺-ATPase Activity by OH and Protection by ATP
When purified cardiac (35 µg/mL) and skeletal muscle (14 µg/mL) SR vesicles were exposed to various concentrations of the hydroxyl free radical (·OH)–generating system, a dose-dependent inhibition of Ca²⁺-ATPase enzymatic activity was seen for both cardiac and skeletal muscle SR (Fig 1). Complete inhibition of cardiac Ca²⁺-ATPase occurred when the SR was exposed to 1 mmol/L H₂O₂+0.1 mmol/L Fe³⁺-NTA. Complete inhibition of skeletal muscle Ca²⁺-ATPase occurred with exposure to 1 mmol/L H₂O₂+0.2 mmol/L Fe³⁺-NTA. Neither H₂O₂ (1 mmol/L) nor Fe³⁺-NTA (0.2 mmol/L) alone had any effect on enzymatic activity (data not shown). The singlet oxygen scavenger histidine (1 mmol/L)²¹ did not prevent denaturation of the enzyme, whereas the ·OH scavenger mannitol (50 mmol/L) resulted in complete protection (data not shown), further confirming that the loss of enzyme activity was due to ·OH-mediated attack rather than secondary to singlet oxygen formation. When the same amount of purified enzyme was premixed with 1 mmol/L ATP before exposure to the ·OH-generating system (1 mmol/L H₂O₂+0.2 mmol/L Fe³⁺-NTA), significant protection of both cardiac and skeletal muscle SR Ca²⁺-ATPase was observed (Fig 1). No protection occurred when the enzymes were premixed with adenosine, sucrose, AMP, or ADP (1 mmol/L each) before exposure of the enzyme to the same ·OH free radical–generating system. A small amount of protection occurred with AMP or ADP when the vesicles were exposed to lower concentrations of ·OH. ATP protected SR Ca²⁺-ATPase activity in a dose-dependent fashion (Fig 2): 65% of the enzymatic activity of cardiac SR Ca²⁺-ATPase was conserved in the presence of 100 µmol/L ATP, whereas only 14%, 15%, 21%, and 27% of enzymatic activity was maintained for 10 nmol/L, 100 nmol/L, 1 µmol/L, and 10 µmol/L ATP, respectively. Complete preservation of enzymatic activity was seen in the presence of 1 mmol/L ATP.

View larger version (13K): [in this window] [in a new window]   Figure 2. Dose-dependent ATP protection of the cardiac SR Ca2+-ATPase. Purified cardiac Ca2+-ATPase (10 µg/mL) was incubated with various concentrations of ATP (0.01 µmol/L, 0.1 µmol/L, 1 µmol/L, 10 µmol/L, 100 µmol/L, 1 mmol/L, and 10 mmol/L) in the presence or absence of the ·OH-generating system (0.2 mmol/L Fe3+-NTA+1 mmol/L H2O2), as described in "Materials and Methods." The data represent the mean of four independent experiments.

Effect of ·OH on SR ⁴⁵Ca Uptake
Complete inhibition of Tg-sensitive ⁴⁵Ca uptake occurred after exposure to ·OH in the absence of ATP. No significant protection of ⁴⁵Ca uptake was seen in the presence of AMP (1 mmol/L) or ADP (1 mmol/L) for both cardiac and skeletal muscle SR Ca²⁺-ATPase. In contrast, complete protection was observed when both types of SR were premixed with 1 mmol/L ATP, as shown in Fig 3.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of AMP, ADP, and ATP on the inactivation of 45Ca uptake of cardiac and skeletal muscle SR Ca2+-ATPase by hydroxyl free radical. Purified cardiac (0.28 mg/mL) and skeletal (0.29 mg) muscle Ca2+-ATPase were incubated with the hydroxyl free radical–generating system (Fe3+-NTA+H2O2) in the presence of either AMP, ADP, or ATP (1 mmol/L each), as described in "Materials and Methods." Each bar represents various conditions as indicated. Values are means of three independent experiments. Experimental data show that 45Ca uptake of both cardiac and skeletal muscle SR Ca2+-ATPase was protected by 1 mmol/L ATP in the presence of the ·OH-generating system but not by 1 mmol/L ADP and AMP.

Time Course of ·OH Radical Inhibition
After addition of the ·OH-generating system (0.2 mmol/L Fe³⁺-NTA+1 mmol/L H₂O₂) to cardiac SR, 70%, 80%, and 95% inhibition of Ca²⁺-ATPase activity was reached at 5, 10, and 15 minutes, respectively (Fig 4). The enzymatic activity of the cardiac SR Ca²⁺-ATPase was completely destroyed at 25 minutes.

View larger version (14K): [in this window] [in a new window]   Figure 4. Time course of the ·OH radical inhibition of the cardiac SR Ca2+-ATPase. Purified cardiac Ca2+-ATPase (22 µg/mL) was incubated in the presence of ·OH free radical (0.2 mmol/L Fe3+-NTA+1 mmol/L H2O2) at room temperature for 5, 10, 15, 20, 25, and 30 minutes as indicated. Each sample was then assayed for standard Tg-sensitive ATPase activity (see details in "Materials and Methods") in the presence of 1 mmol/L ATP and compared with the control (without the ·OH-generating system). The results represent the mean of three experiments.

Effect of ·OH on Primary Structure of Ca²⁺-ATPase
To investigate whether the concentration of ·OH used in our experiments damaged the polypeptide chain of the Ca²⁺-ATPase, purified cardiac SR (44 µg/mL) was incubated with 1 mmol/L H₂O₂+0.2 mmol/L Fe³⁺-NTA. PAGE demonstrated no change in the relative amounts of the 97-kD band or in the apparent molecular weight of the Ca²⁺-ATPase polypeptide, and no tattered peptide fragments were observed (Fig 5). This is in contrast to the loss of the 97-kD band reported to occur as a result of singlet oxygen.

View larger version (59K): [in this window] [in a new window]   Figure 5. Effect of ·OH free radical on the primary structure of the cardiac SR Ca2+-ATPase. Purified enzyme (20 µg/mL) was incubated with 1 mmol/L H2O2+0.2 mmol/L Fe3+-NTA for 30 minutes and separated by SDS-PAGE. The gel was stained and clarified. Lanes are as follows: A, protein marker; B, control, without ·OH; and C, ·OH-treated sample. Note that ·OH radicals do not have a significant effect on the 97-kD band of Ca2+-ATPase.

Lack of Scavenging of ·OH by Adenine Nucleotides
To investigate whether ATP, in the concentration used in the present study, acts as a direct scavenger of ·OH, EPR measurements were performed in the presence of the spin trap DMPO (100 mmol/L) and quantified by double integration. No significant signal was detected with buffer alone, but in the presence of ·OH (1 mmol/L H₂O₂+0.2 mmol/L Fe³⁺-NTA), a prominent EPR signal was seen consisting of a quartet (1:2:2:1) signal with hyperfine coupling constants of nitrogen (14.9 Gauss) and hydrogen (14.9 Gauss) indicative of DMPO-OH (Fig 6).²² This signal was not altered by ATP, ADP, or AMP (Fig 6). In addition, the time course and maximal level of ·OH generation were not altered by ATP, ADP, or AMP (Table).

View larger version (19K): [in this window] [in a new window]   Figure 6. EPR spectra of ·OH free radical–generating system in the presence of 100 mmol/L DMPO under various conditions. Spectra are as follows (from top to bottom): 15 mmol/L imidazole/HCl buffer as background, H2O2+Fe3+-NTA as control, H2O2+Fe3+-NTA+ATP, H2O2+Fe3+-NTA+ADP, and H2O2+Fe3+-NTA+AMP. The final concentrations of H2O2, ATP, ADP, and AMP were 1 mmol/L each, and Fe3+-NTA was 0.2 mmol/L. The DMPO-OH quartet complex signal of each sample was chosen at the maximal ·OH generation level. The ·OH generation rate constant was calculated as shown in the Table.

View this table: [in this window] [in a new window]   Table 1. Kinetics of ·OH Generation From 1 mmol/L H2O2+0.2 mmol/L Fe3+-NTA in the Presence of ATP, ADP, or AMP (1 mmol/L Each)

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oxygen-derived free radicals are produced in the heart after ischemia/reperfusion and are believed to cause widespread peroxidation of lipid membranes. In addition, alteration of membrane proteins by free radicals probably also represents an important factor in the evolution of myocardial ischemia/reperfusion damage. However, the free radical–targeting sites and the derangements responsible for the altered function of key enzymes, such as the Ca²⁺-ATPase, which regulates Ca²⁺ homeostasis in heart muscle, are not yet fully understood. In the present study, we demonstrate that ·OH generation of a magnitude similar to that observed during the early minutes of postischemic reperfusion is sufficient to denature the Ca²⁺-ATPase. To further investigate whether the active site of the SR Ca²⁺-ATPase is involved in the mechanism of free radical inhibition, ATP was used to specifically protect enzymatic activity against ·OH-induced inactivation. If, in the presence of the specific site protector, free radical–induced inhibition of the protein results in a significantly smaller degree of inactivation than occurred in the absence of the protector (all other conditions being the same), this suggests that, at least in part, the free radical–induced inhibition occurs within the active site. Our results show that the occupation of the active site by ATP protects against ·OH free radical–induced loss of enzymatic activity and Ca²⁺ active transport in both cardiac and skeletal muscle SR, suggesting that the ·OH free radical denatures the SR Ca²⁺-ATPase by directly attacking the ATP binding site of the enzyme, thereby impairing its ATPase activity and its function as the SR Ca²⁺ pump. SDS–gel electrophoresis did not show evidence of primary structural damage to the enzyme by ·OH, supporting the concept that ·OH inhibits the Ca²⁺-ATPase by producing a targeted lesion at a specific critical site. However, the ·OH radical could produce tertiary structure changes at the ATP binding site and in sites other than the active site of the Ca²⁺-ATPase.

Ca²⁺ uptake by the SR is an energy-dependent process mediated by the hydrolysis of ATP, which is catalyzed by the Ca²⁺-ATPase. Once cellular energy stores are depleted, Ca²⁺ uptake by the SR no longer serves as a Ca²⁺ sink. It has been previously reported that myocardial ATP declines by 35% to 50% within the first 15 minutes after ischemia or anoxia in open-chest dogs and that ATP levels fall to <10% after 40 minutes.^{22 23 24 25 26} Reperfusion or reoxygenation after a sufficiently long period of ischemia/anoxia results in a rise in cytoplasmic Ca²⁺ concentration, presumably due to an inability of the SR to transport and sequester sufficient Ca²⁺ quickly enough. ATP generated by glycolysis may be functionally coupled to the SR Ca²⁺-ATPase during the critical transition phase from hypoxia to normoxia.¹⁴ The resulting rise in cytosolic Ca²⁺ may lead to cellular injury through activation of Ca²⁺-dependent cellular proteases and lipases.⁵

It is unknown why SR Ca²⁺ transport is inadequate under these conditions. One possibility is that cellular ATP levels decline to the point where the Ca²⁺-ATPase cannot function. However, the K_m of rabbit cardiac SR Ca²⁺-ATPase for ATP is 0.22 mmol/L (data not shown), which is lower than the mean cellular levels measured after ischemia or hypoxia. On the other hand, the concentration of ATP at the active site of Ca²⁺-ATPase is unknown and could be much lower than the mean cellular level.

A second possibility is that loss of SR function could occur because of the direct attack by free radicals generated at the time of reperfusion or reoxygenation on the SR Ca²⁺-ATPase. Our results suggest that this attack may be targeted at the ATP binding site of the enzyme and result in loss of active transport of Ca²⁺ into the SR. An interesting possibility is that cellular ATP depletion may amplify free radical–induced damage to the Ca²⁺-ATPase. Occupation of the ATP binding site by ATP protected the enzyme against ·OH-induced loss of function, suggesting that ATP depletion might accelerate the irreversible denaturation of the enzyme. It has been previously shown that the ·OH-generating system used here, infused into the isolated rabbit heart, results in a similar magnitude of radical generation as that seen upon reperfusion.¹⁶ Thus, it is possible that the reperfusion burst of radical generation could be sufficient to inactivate the SR Ca²⁺-ATPase in a manner similar to that observed in the present study. Moreover, ATP protects SR Ca²⁺ active transport in the presence of the ·OH radical (Fig 2), further demonstrating that ATP depletion may be an important factor in the mechanism of the free radical–induced myocardial injury.

It has been reported that lipid peroxidation is a hallmark of ischemic/reperfusion injury.^{27 28 29} The inhibition of the cardiac SR Ca²⁺-ATPase observed after ischemia/reperfusion could be related, in principle, to changes in the lipid structure of the SR membrane. However, our ⁴⁵Ca uptake experiments showed that after exposure of SR vesicles to ·OH, ion transport activity of the membrane-bound SR Ca²⁺-ATPase remained completely intact in the presence of l mmol/L ATP. This suggests that OH-induced inhibition of SR Ca²⁺ transport is related to damage to the Ca²⁺-ATPase itself rather than to peroxidation of the surrounding SR lipid membrane, with secondary effects on enzyme function.

Although AMP and ADP share a major part of the chemical structure of ATP, this structural similarity failed to significantly preserve the enzymatic activity of the protein or the ⁴⁵Ca uptake of cardiac and skeletal muscle SR in the presence of ·OH, presumably because of the lack of high-affinity binding to the active site of the Ca²⁺-ATPase. The fact that only ATP protected the SR Ca²⁺-ATPase against OH-induced denaturation suggests that only the binding of ATP to the SR Ca²⁺-ATPase leads to the formation of a phosphorylated intermediate, as described earlier,^{30 31} and suggests that this conformational state of the SR Ca²⁺-ATPase may play a crucial role in protecting certain essential amino acids located at the ATP binding site.

Proteins containing the amino acids tryptophan, tyrosine, phenylalanine, histidine, methionine, and cysteine can undergo free radical–mediated amino acid modification by oxidation/reduction reactions in a neutral aqueous milieu.^{32 33} Under our experimental conditions, ·OH free radicals probably react with various amino acids all around the enzyme. However, the results indicate that ·OH denatures SR Ca²⁺-ATPase by attack on vital residues within the active site of the enzyme that account for the biological activity rather than by attack elsewhere on the molecule. It has been shown that aspartate₃₅₁ within the active site accepts the phosphate of ATP during the first step of its hydrolysis.^{34 35 36} Since oxidation of sulfhydryl groups of the protein represents a likely mechanism of free radical damage,^{32 33} we hypothesize that cysteine₃₄₉, adjacent to aspartate_35l, is likely to be involved. Although it is not certain how many essential amino acids constitute the three-dimensional structure of the active site of the Ca²⁺-ATPase and whether cysteine₃₄₉ contributes to ATP binding, it is clear that the cysteine₃₄₉ is located near the phosphorylation site of the enzyme and, therefore, could be protected from free radical attack by a conformational change of the ATP binding site during phosphorylation. We do not know how many essential amino acids can be protected by such a conformational change of the Ca²⁺-ATPase, but our results clearly show that the binding of ATP to the enzyme before exposure to ·OH protects a critical portion of the ATP binding site.

More detailed investigations of the specific amino acids involved will increase our understanding of the pathophysiology of myocardial injury during ischemia/reperfusion and may help to provide the basis for a more rational therapeutic approach for optimal protection of the heart.

	Selected Abbreviations and Acronyms

 

DMPO = 5,5-dimethyl-1-pyrroline N-oxide EPR = electron paramagnetic resonance NTA = nitrilotriacetic acid SR = sarcoplasmic reticulum Tg = thapsigargin

	Acknowledgments

 
This study was supported by National Institute of Health grants HL-33360, HL-52315, and HL-38324 and Johns Hopkins institutional research grant S07 RR05378. We thank Penghai Wang for assisting us in preparation of the cardiac and skeletal muscle SR Ca²⁺-ATPase and Dr P. Kuppusamy for supervision of the technical aspects of the electron paramagnetic resonance measurements and the analysis of the kinetic data.

Received August 23, 1996; accepted October 2, 1996.

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