Animal studies.

The experiments described below were approved by the University of California Davis Animal Care and Use Committee. Male Wistar rats (of various ages as indicated) were purchased from a commercial supplier (Charles River Laboratories, Wilmington, MA). All cages were kept in a vivarium maintained at 22 ± 1°C, with a 12:12-h light-dark cycle, following National Institutes of Health guidelines for animal care and use. The lights were set to go out at 1900. Cage maintenance was conducted during the light cycle. Briefly, after arrival in our laboratory, rats were fed a commercially available diet and allowed a few days to relieve any stress that they may have incurred during the travel and get acquainted with the new location. Over the testing period, the rats had free access to their food 24 h/day, except when cage maintenance was conducted. On the 3rd or 4th day, the rats were removed from their cages and taken to the surgery room, where they were euthanized with CO₂. Rats were euthanized between 9 and 10 AM. The livers were quickly removed from the rats, their wet weights were recorded, and they were rapidly homogenized for mitochondria isolation.

Isolation and subfractionation of rat liver mitochondria.

Percoll-purified mitochondria and their fractions were isolated from male Wistar rats, as described before (31).

Preparation of phosphorylating submitochondrial particles.

The subfractionation of mitochondria was accomplished by subsequent treatments with digitonin and sonication, as described previously (62). Submitochondrial particles (SMP) were prepared by a procedure similar to that described by Pedersen and Hullihen (56) with the following minor modifications. Mitoplasts (mitochondria with the OM removed) were prepared by adding digitonin to the mitochondrial suspension, prepared as described before. The mitoplasts were disrupted by sonication and the SMP collected from a centrifugation step (62). The inverted orientation of the inner mitochondrial membrane in SMP was determined from the rate of oxygen uptake in the presence of 10 mM succinate and stimulated by 100 μM cytochrome c essentially as described by Guerrieri and Papa (34). Under our experimental conditions, the percentage of inversion was found to be >95%. Since rat liver mitochondria are known to contain an ATPase inhibitor peptide [or IF₁ (58)], it remained possible that SMP prepared as described above may contain a large population of IF₁-inhibited ATPase molecules. To obtain SMP with low IF₁, a final step that essentially followed the procedure described by Racker and Horstman (60) was incorporated with the following modifications. The step involving the chromatography through Sephadex G-50 was replaced by concentrating and purifying the SMP in Centricon tubes, with a nominal molecular mass cut off of 30 kDa. The retentate was washed three times with buffer (2 mM EDTA, 75 mM sucrose, 250 mM KCl, and 30 mM HEPES, pH 8.0) at room temperature. By doing this procedure, the ATPase activity increased by three to five times. SMP were characterized for contamination with other submitochondrial compartments by evaluating the activities of monoamine oxidase (OM), adenylate kinase (IMS), and malate dehydrogenase (M), as described before (25); the contamination with mitochondrial components present at compartments other than the IM was 2.3, 3.3, and 7.6%, respectively, of the activities present in the original rat liver mitochondria preparation.

Preparation of F₁-deficient SMP and F₁-enriched fractions and procedure for reconstituting F₁ with F₁-deficient SMP.

SMP were treated with urea to remove the F₁ portion of the ATP synthase (56). These urea-treated particles were devoid of F₁ but still contained the membrane sector subunits and the interface subunits of the ATP synthase. Residual ATPase activity of F₁-stripped SMP, evaluated as described in Enzymatic assays, was always <10%, as described by others (56). The uncoupling agent FCCP, at concentrations that markedly stimulated the ATPase activity of intact mitochondria (10 μM), had no significant effect on the ATPase activity of urea particles (55). In addition, concentrating and purifying these urea particles through Centricon (NMWL = 30 kDa) was without a significant effect on the residual ATPase activity. These experiments showed that most of the SMP had no detectable IF₁-inhibited ATPase. To monitor that the treatment with urea removed F₁ but did not damage or alter the remaining F_o-bound SMP, the rate of oxygen consumption by F₁-stripped SMP sustained by succinate (and inhibited by 3 μg/ml oligomycin) was followed as described by Pedersen and Hullihen (55).

The F₁ portion was prepared from coupled [with respiratory control ratios of ≥5 (17)] mitochondria, using the procedure originally described by Catterall and Pedersen (15, 16). The F₁ particles prepared by using this method were then separated by DEAE chromatography followed by Sephadex G-25. The different fractions were analyzed in parallel dot blots, utilizing antibodies against nitrotyrosine and β-subunit. The fractions that had the highest nitration level per area of β-subunit as indicated by the optical density ratio of nitrated Tyr/total β-subunit were pooled and lyophilized immediately. Reconstitution of F₁ with the urea-treated particles was achieved using the procedure described in detail by Pedersen and Hullihen (56).

Immunoprecipitation procedure.

Mitochondrial fractions [T (total mitochondria), IM, and CS], each containing 100 μg of protein, were incubated for 1 h with either 2.5 μg/ml of antibody against ATPase β-subunit (catalog no. 612518; BD Biosciences) or 50 μg/ml of antibody against nitrotyrosine (mouse antibody, catalog no. 05-233; Upstate Biotechnology). Each of these samples (100 μl each) was then mixed with 0.9 ml of immunoprecipitation (IP) buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 0.5% NP-40] and 0.5% vol/vol of 10% protein A-agarose. The samples were then incubated for 1 h and centrifuged, and the pellet was washed three times with IP buffer. The pellets were resuspended in 50 μl of 2× Laemmli buffer, boiled for 5 min, and centrifuged, with the supernatants subsequently cooled on ice. The immunoprecipitated proteins were separated using 10% SDS-PAGE. Gels were used for Western blots of β-subunit or nitrotyrosine essentially as described below, except that blocking was carried out with either 1% Carnation nonfat dry milk (NFDM) or 1% NFDM supplemented with 250 μg/ml of mouse F(Ab)₂ where indicated.

Evaluation of protein nitration under conditions of endogenous nitric oxide production.

Percoll-purified coupled rat liver mitochondria (1 mg protein/sample) were incubated with 0.1 mM l-arginine or N^G-monomethyl-l-arginine (NMMA) and a respiratory substrate in an incubation medium containing calcium in amounts shown to be sufficient to elicit sustained production of nitric oxide (65). At each time point (0, 30, 60, and 120 min), mitochondria were pelleted and washed extensively to remove remaining l-arginine or NMMA, and the mitochondrial proteins were analyzed using two-dimensional (2D) gels (25). The gels were stained with SYPRO Ruby (Molecular Probes), and parallel ones were blotted and probed for nitrotyrosine as described for Western blots below. The spot on the 2D gels that was identified as the β-subunit of F_oF₁-ATPase using the respective antibody was excised, trypsinized in situ, and analyzed by matrix-assisted laser desorption/ionization time of flight [MALDI-TOF; as explained before; (25)] at the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, Twin Cities. To follow nitric oxide production, we performed two parallel assays, the NMMA-sensitive oxidation of oxymyoglobin in the presence of nitric oxide to yield metmyoglobin and the scintillation proximity assay by using beads labeled with l-arginine. The reaction medium for the former assay contained substrate (either malate-glutamate or succinate) and 0.1 mM oxymyoglobin. Oxymyoglobin was prepared as described previously (29). The reaction was monitored at 581–592 nm at 22°C, as described previously (29). For the latter assay, the activity of mitochondrial nitric oxide synthase was monitored using the scintillation proximity assay. Essentially, nitric oxide production was determined through the conversion of l-[³H]arginine to l-[³H]citrulline in the previous medium with mitochondria, l-arginine, and substrate, to which 0.1 μM l-[³H]arginine was added. Aliquots were taken at various time points, and the reaction was stopped by adding 1 mg of SPA Arg-binding beads (Amersham Biosciences) in 50 mM NaOH. Beads were allowed to settle for 2 h, and then the remaining amount of l-[³H]arginine in the reaction was determined by using the Yttrium Silicate SPA in conjunction with the Beckman 1450 MicroBeta plate counter.

2D gel electrophoresis.

Mitochondrial fractions were separated by pI on precast gel strips (IPG strips) with a linear gradient ranging from pH 3 to 10, using the Multiphor II with the Immobiline Strip tray and following the manufacturer's instructions (Amersham Pharmacia Biotech, Piscataway, NJ). The IPG strips were rehydrated overnight in rehydration buffer (6 M urea, 2 M thiourea, 2% NP-40, 2% IPG buffer, pH 3–10, 0.1 M dithiothreitol) prior to use. Twenty micrograms of mitochondrial sample was solubilized in the rehydration buffer for 30 min at room temperature prior to being separated, using the rehydrated strips. The voltage was provided by a Hoefer power supply PS 3501 XL, and the program utilized was as follows: 300 V for 1 min, followed by a linear gradient from 300 to 3,500 V for 1.5 h, then fixed voltage at 3,500 V for 4.5 h. The strips were preequilibrated with 6 M urea, 30% glycerol, 1% SDS, 5 mg/ml dithiothreitol, and 50 mM Tris, pH 6.8, for 10 min at room temperature prior to the samples being separated in the second dimension using SDS-PAGE; the 10% gel was run in a Hoefer SE600 overnight at 50 V. Gels were electroblotted and analyzed using antibodies against nitrotyrosine and the β-subunit of F_oF₁-ATPase using standard Western blot procedures (see below).

Enzymatic assays.

ATP synthesis was detected spectrophotometrically at 340 nm and 22°C by coupling the production of ATP to the reduction of NADP⁺ via the hexokinase and glucose-6-phosphate dehydrogenase reactions (57, 59). The assay mixture, in a final volume of 1 ml, contained 1 mM ADP, 10 mM potassium phosphate, pH 7.5, 10 mM succinate, 50 mM HEPES, 1 mM glucose, 1 mM NADP⁺, 2 mM MgCl₂, 2 mg/ml fatty acid-free BSA, 0.2 M sucrose, 20 IU hexokinase, and 7.0 IU glucose-6-phosphate dehydrogenase. The reaction was initiated by addition of mitochondrial protein from 50 to 200 μg of SMP to ensure linear dependency of activity with protein. The rate of NADPH production was followed for 1–3 min, and then 5 μg/ml oligomycin was added and the reaction followed for another 2 min. The rate of NADPH production sensitive to oligomycin was taken as the rate of ATP synthesis.

ATPase activity was measured spectrophotometrically at 340 nm by coupling the production of ADP to the oxidation of NADH via the pyruvate kinase and lactic dehydrogenase reactions, as described previously (51). The reaction mixture, in a volume of 1 ml at pH 7.5 and 22°C, contained 4 mM ATP, 4.8 mM MgCl₂, 0.40 mM NADH, 0.6 mM phosphoenolpyruvate, 5 mM potassium cyanide (KCN), 1 IU lactic dehydrogenase, 1 IU pyruvate kinase, 2.5 mM potassium phosphate, 80 mM HEPES, and 10–60 μg of SMP. Initial rates sensitive to oligomycin inhibition were used in all specific activity calculations (57). ATPase activity was also determined using a microplate approach, as described previously (27).

Western blots.

SDS-PAGE was performed using 10% gels. The gels were electroblotted and treated with monoclonal antibodies against nitrotyrosine (Upstate Biotechnology) or with serum raised in rabbits against the rat liver β-ATPase (kind gift from Dr. J. Cuezva, Universidad Autónoma de Madrid, Spain), as described previously (25, 27). To assure specificity for nitrotyrosine, blots were blocked with 10 mM nitrotyrosine or the mitochondrial samples were pretreated with 10 mM sodium persulfate (Na₂S₂O₈) or ascorbic acid to reduce nitrotyrosine to aminotyrosine, a molecule not recognized by the antibody (Supplemental Fig. S1; Supplemental Material for this article can be found at the AJP-Endocrinology and Metabolism web site). The immunocomplexes were developed using an enhanced horseradish peroxidase-luminol chemiluminescence reaction (Amersham) and detected using photographic film (Hyperfilm ECL). The level of nitration was evaluated by processing the spots with the KodakImager 2000MMM and using the software provided by the manufacturer. The original gels were loaded with varying amounts of protein to ensure a linear response between chemiluminescence and protein. The specific nitration (defined as the nmol of nitrotyrosine/g protein) was determined using semiquantitative dot blots and nitrated BSA as a standard. The nitration of peroxynitrite-treated BSA was evaluated by dialyzing the protein after exposure to peroxynitrite, followed by acid hydrolysis, and evaluating the content of nitrotyrosine by using high-performance liquid chromatography (HPLC) with diode array and electrochemical detections. Protein was quantified using the Lowry assay and BSA as a standard (45).

HPLC with fluorescence detection for evaluation of nitrotyrosine in mitochondria.

HPLC samples were prepared by digesting mitochondria in the presence of proteinase K [1:20 (wt/wt), enzyme-protein] in 0.2 M phosphate buffer (pH 7.4) for 2–3 h at 50°C and then treating with a second equal aliquot of the protease and incubating for an additional 12–16 h. Following digestion, samples were divided into three parts. One was left untreated, the second was treated with 60 mM sodium dithionite to reduce nitrotyrosine to aminotyrosine, and the third was also reduced with 60 mM sodium dithionite and spiked with a known amount (10 pmol) of aminotyrosine. The digested samples were analyzed by reversed-phase HPLC using two C18 columns (4.6 × 250 mm) in line. A linear gradient was maintained at a flow rate of 0.8 ml/min as follows: from 100 to 98% mobile phase A (A) in 10 min, 98 to 50% A in 20 min, 50 to 25% A in 25 min, and then from 25 to 0% A in 40 min. Mobile phase A consisted of 50 mM sodium citrate and 50 mM acetic acid (pH 3.1), whereas mobile phase B was 10% methanol, 50 mM sodium citrate, and 50 mM acetic acid (pH 3.1). The eluted fractions were monitored with a diode array and fluorescence detectors (excitation and emission wavelengths 277 and 307 nm, respectively). The aminotyrosine quantification was performed by measuring the peak area and calculating its amount from a standard curve (ensured to be linear). The limit of detection using this method was <3 pmol/mg mitochondrial protein. Nitrated BSA was evaluated using the same method as for mitochondrial proteins.

As a control to ensure efficient proteolysis of mitochondrial proteins, azocasein hydrolysis was measured by following the absorbance of the azo dye at 390 nm. Proteolytic activity was evaluated using 1 mg/ml azocasein (Sigma Chemical) incubated with 1 mg/ml of mitochondrial protein that had been digested with proteinase K, as described above. Upon completion of proteolysis (when no significant changes in absorption at 390 nm were obtained), 10% trichloroacetic acid was added to the hydrolysate and samples were centrifuged for 10 min at 12,000 g. The supernatants were analyzed by following the absorbance at 390 nm. Absorbance measurements were compared with those of azocasein digested alone (100% digestion) and azocasein without proteinase K (0% proteolysis).

Impact of nitrative stress on ATPase activity.

The F₁-ATPases contain six nucleotide-binding sites in the beef heart enzyme. Three of these sites bind and exchange nucleotides readily during catalysis (19, 21), suggesting a direct role in enzyme activity. In contrast, the three other sites, once filled with adenine nucleotides, exchange very slowly during turnover and are therefore called noncatalytic. Chemical modification studies with 2-azido-ATP have provided evidence that Tyr³⁴⁵ of the F₁ β-subunit is located at catalytic sites of the beef enzyme, whereas Tyr³⁶⁸ is located at noncatalytic sites (20). Given the observed nitration of the rat β-subunit in vivo and in vitro, we investigated the effect of nitration on F_oF₁-ATPase activity by measuring ATP synthesis and hydrolysis rates through the use of coupled enzyme assays. These activities were evaluated in reconstituted samples consisting of either control or nitrated F₁ with F₁-deficient particles (or urea particles) from rat liver. The nitrated F₁ particles were obtained from l-arginine-supplemented rat liver mitochondria incubated for 60 min, separated, and collected by chromatography. Control F₁ particles were not subjected to l-arginine but were purified in a similar manner. Reconstitution of F₁ with urea particles (also obtained from rat liver) provided the means for evaluating the sole effect of F₁ subunits on the ATPase. Our results indicated that there was a reciprocal correlation between Tyr nitration of the β-subunit and the ATP hydrolysis and synthesis rates (Table 2); that is, the more nitration, the lower the rates. Since mass spectrometry does not provide quantitative information on the extent of nitration at each residue, we cannot comment on the relative contribution of each nitrated Tyr residue toward decreasing the synthesis and hydrolysis activities, only that both Tyr³⁴⁵ and Tyr³⁶⁸ were nitrated to some extent.

As mentioned in the previous section, in addition to the Tyr nitration, we also detected Met sulfoxide formation from mitochondria subjected to nitrative stress. Chemical modification to Met residues can be a double-edged sword, sometimes inducing conformational changes causing loss of protein activity (8, 9), whereas other modifications can have a crucial effect on protein interaction without large conformational changes or, at other times, protecting the protein by acting as an antioxidant (66). Nevertheless, considering that Met sulfoxide has been implicated in activity loss and the fact that oxidized Met residues detected (see Table 1) are located at conserved regions in the β-subunit, it could be speculated that these residues may have contributed to the activity loss. However, there are two observations that undermine this argument. 1) Substitution of Met with a slightly more polar amino acid, such as its sulfoxide derivative, does not substantially change the hydropathy index,² and 2), most importantly, a double-point mutation (Y345F/Y368F) of the β-subunit completely preserved ATPase activity under nitrative stress in vitro (27). Similar experiments conducted on wild-type β-subunit documented ~34% residual ATPase activity (28), a value that agrees well with the residual ATPase activity under nitrative stress reported in this study (~27%; Table 2). These arguments suggest that the Met sulfoxide formation is not the main cause for decreased ATPase activity during nitrative stress.

Role of nitrated β-subunit in ATPase activity under in vivo conditions.

The experiments depicted above ascertained an effect of nitrated Tyr on ATPase activity but did not provide any information as to whether this effect was relevant to the animal under physiological and pathological conditions. To investigate this possibility, the level of nitration and the ATPase activities of SMP from animals of various ages, namely young (4 wk old), adult (16 wk old), and old rats (80 wk old), were evaluated. As demonstrated previously using mass spectrometry (see above), nitration of Tyr³⁶⁸ was evident in young rats, and nitration was predominantly of Tyr³⁶⁸ in adult rats, albeit with a minor contribution of nitration of Tyr³⁴⁵. In older animals, the nitration of both residues was significant. In experiments carried out under the same conditions, a decline in enzymatic activity, V_max, was observed to accompany the increase in the nitration of the β-subunit (Table 3). These results corroborate the conclusions obtained previously with an in vitro system (27); nitration of Tyr³⁶⁸ and/or Tyr³⁴⁵ decreases the activity of ATPase, suggesting that maintenance of these residues in a nonnitrated state is critical to the full biological activity of this enzyme.

To provide a more complete understanding of how nitration of these Tyr residues leads to decreased ATPase activity, we performed kinetic studies on the ATPase from rats of different ages. To this end, the initial rates for ATP hydrolysis for each group were tested in the presence of 10–2,000 μM ATP (Fig. 3). Woolf-Augustinsson-Hofstee plots of the kinetic data resulted in parallel lines, indicating that the nitration of Tyr residues resulted in an effect similar to that observed with an irreversible, noncompetitive inhibitor. This suggests that, although nitration had no effect on substrate affinity (similar K_m values; Table 3), nitration at either Tyr site created a substantial alteration to the ATPase structure that resulted in decreasing V_max by a factor of two (Fig. 3 and Table 3). In agreement with this possibility, we have shown significant changes in the structure of ATPase as a result of nitration by modeling X-ray crystallography structures of F₁ (27).

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Fig. 3.

Activity of liver ATPase in young, adult, and old animals. The activity of ATPase was evaluated (following the procedure described in experimental procedures) in rat liver mitochondria from young (▲), adult (○), and old rats (●).