Gene 286 (2002) 135–141 www.elsevier.com/locate/gene
Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage Giuseppe Paradies*, Giuseppe Petrosillo, Marilva Pistolese, Francesca Maria Ruggiero Department of Biochemistry and Molecular Biology and C.N.R. Unit for the Study of Mitochondria and Bioenergetics, University of Bari, VIA E. Oraboni 4, 70126 Bari, Italy Received 13 July 2001; received in revised form 18 October 2001; accepted 7 November 2001 Received by M.N. Gadaleta
Abstract The aim of this study was to investigate the influence of reactive oxygen species (ROS) on the activity of complex I and on the cardiolipin content in bovine heart submitochondrial particles (SMP). ROS were generated through the use of xanthine/xanthine oxidase (X/XO) system. Treatment of SMP with X/XO resulted in a large production of superoxide anion, detected by acetylated cytochrome c method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to ROS generation resulted in a marked loss of complex I activity and to parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD 1 catalase. Exogenous added cardiolipin was able to almost completely restore the ROS-induced loss of complex I activity. No restoration was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, nor with peroxidized cardiolipin. These results demonstrate that ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those pathophysiological conditions characterized by an increase in the basal production of reactive oxygen species such as aging, ischemia/reperfusion and chronic degenerative diseases. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Reactive oxygen species; Complex I; Cardiolipin; Beef heart submitochondrial particles
1. Introduction Oxygen free radicals are highly reactive species which are known to be the major factor in oxidative cell injury via the oxidation and subsequent functional impairment of lipids, proteins and nucleic acids. The cellular and subcellular targets of free radical attack and the metabolic consequences of the resulting molecular alterations have not been established. Mitochondrial electron transport chain is considered as a major intracellular source of reactive oxygen species (ROS), mainly at the level of the NADH dehydrogenase and at the level of coenzyme Q (Chance et al., 1979; Turrens and Boveris, 1980; Cadenas et al., 1977). The superoxide anion appears to be the first oxygen reaction product generated under physiological and pathological conditions. Subsequent dismutation of superoxide anion generate H2O2 which Abbreviations: SMP, submitochondrial particles; NAO, 10-N-nonyl-lacridine orange; ROS, reactive oxygen species; SOD, superoxide dismutase; HPLC, high-pressure liquid chromatography; X/XO, xanthine/ xanthine oxidase * Corresponding author. Fax: 139-80-544-3317. E-mail address:
[email protected] (G. Paradies).
in turn can lead to production of zOH. It has been estimated that during state 4 respiration isolated mitochondria generate approximately 1 nmol hydrogen peroxide per mg of protein, which accounts for approximately 2% of total mitochondrial oxygen uptake (Boveris and Chance, 1973). Mitochondrial membrane constituents are particularly vulnerable to oxidative damage by oxygen free radicals which are generated continuously by the mitochondrial respiratory chain. Peroxidation of membrane lipids components has been hypothesized to be a major mechanism of oxygen free radicals attack resulting in generalized impairment of the membrane function. The possibility that such a mechanism could cause specific damage to certain vital components of the mitochondrial membrane deserves further attention. Due to its membrane composition, the mitochondrion is especially sensitive to lipid peroxidation. In fact, the major phospholipid components of the mitochondrial membranes are rich in unsaturated fatty acids that are particularly susceptible to oxygen radical attack because of the presence of double bonds, that can undergo peroxidation through a chain of oxidative reactions. Cardiolipin, a phospholipid of unusual structure localized
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00814-9
136
G. Paradies et al. / Gene 286 (2002) 135–141
almost exclusively within the inner mitochondrial membrane, is particularly rich in unsaturated fatty acids (90% represented by linoleic acid). This phospholipid plays an important role in the mitochondrial bioenergetics optimizing the activity of key mitochondrial inner membrane proteins, including several anion carriers and some electron transport complexes (Hoch, 1992; Robinson, 1993). Mitochondrial cardiolipin molecules are possible targets of oxygen free radical attack, due to their high content of unsaturated fatty acids and because of their location in the inner mitochondrial membrane near to the site of ROS production. In this regard, recent results from this laboratory have demonstrated that mitochondrial mediated ROS generation affects the activity of complex III and IV via peroxidation of cardiolipin, due to oxyradical attack to double bonds of its fatty acid constituents (Paradies et al., 1998, 2000, 2001). These results have been useful to explain the molecular basis of the decline in the cytochrome c oxidase activity observed in mitochondria isolated from animal in certain pathophysiological conditions such as aging (Paradies et al., 1993a, 1994a, 1997a) and ischemia/ reperfusion (Paradies et al., 1999), which are characterized by an increase in the basal rate of the ROS production. NADH–ubiquinone oxidoreductase, also known as complex I, is a multisubunit integral membrane complex of the mitochondrial electron transport chain which catalyzes electron transfer from NADH to ubiquinone. Coupled to the transfer of electrons, protons are vectorially translocated across the mitochondrial inner membrane to establish an electrochemical gradient used for the synthesis of ATP. It has been reported that cardiolipin is specifically required for electron transfer in complex I of the mitochondrial respiratory chain. In fact, the presence of this phospholipid has been shown to be an absolute necessity for regenerating enzymic activity from phospholipid-depleted preparation of complex I (Fry and Green, 1981). Therefore, it would be expected that, similarly to complex III and IV (Paradies et al., 1998, 2000, 2001), ROS-induced peroxidative damage to cardiolipin might affect the activity of complex I as well. To explore this possibility, we studied the effect of ROS, generated by the use of xanthine/xanthine oxidase (X/ XO) system, on the activity of complex I and on the cardiolipin content in beef-heart submitochondrial particles (SMP). The results obtained demonstrate that ROS production affects the activity of complex I as consequence of ROS induced oxidative damage of cardiolipin which is essential for the functioning of this multisubunit enzyme complex.
2. Materials and methods 2.1. Chemicals All chemicals used were commercial products of highest available purity. Acetylated ferricytochrome c, decylubiquinone, reduced nicotinamide adenine dinucleotide (NADH),
antimycin A, bovine heart cardiolipin, superoxide dismutase (SOD) and catalase were purchased from Sigma Chemical Co. 10-N-Nonyl-l-acridine orange (NAO) was purchased from Fluka. 2.2. Submitochondrial particle preparation Beef-heart mitochondria were prepared according to Lo¨ w and Vallin (1963) and stored in 250 mM sucrose, 10 mM Tris (pH 7.4) suspension at 220 8C. Mitochondrial suspension was frozen and thawed for three times and diluted to a concentration of 20–30 mg/ml. Submitochondrial particles were prepared by sonicating the mitochondria for 1 min. with a Branson (model 250) sonifier in ice bath under N2 stream. The sonicated mitochondrial suspension was diluted with an equal volume of 250 mM sucrose, 10 mM Tris (pH 7.4) and centrifuged at 12; 000 £ g for 10 min. The supernatant was decanted and centrifuged at 105; 000 £ g for 50 min. The resulting pellet, consisting of SMP, was washed and suspended in 250 mM sucrose. Proteins were determined by the usual biuret method using bovine serum albumin as standard. 2.3. Generation of superoxide anion The generation of superoxide radical was induced by treatment of SMP with xanthine 1 xanthine oxidase as follows. SMP (1 mg/ml) were incubated in a reaction medium of 50 mM phosphate buffer pH 7.2 at 37 8C. At zero time, 300 mM xanthine and 30 mU xanthine oxidase were added. Aliquots were withdrawn at 60 min for estimating the complex I activity and the cardiolipin content. 2.4. Detection of superoxide anion The production of superoxide anion was determined by the acetylated ferricytochrome c reduction test (Boveris, 1984). About 0.5 mg of SMP, dissolved in 3 ml of reaction medium, as reported above, were supplemented with 300 mM xanthine, 30 mU xanthine oxidase and 10 mM of acetylated ferricytochrome c. The superoxide dependent reduction of acetylated ferricytochrome c was followed spectrophotometrically at 550–540 nm with an HP 8453 diode array spectrophotometer. 2.5. Complex I activity The complex I (NADH–CoQ reductase) activity was measured using the diode array spectrophotometer by following the decrease in absorbance of NADH at 340 nm. The assay mixture contained 3 mM sodium azide, 1.2 mM antimycin A, 50 mM decylubiquinone and 50 mM phosphate buffer, pH 7.2. The SMP sample (50 mg) was added to 3 ml of the assay mixture and the reaction was started by the addition of 60 mM NADH. The activity was calculate using an extinction coefficient of 6.22 mM 21 cm 21 for NADH. The specific activity of the enzyme is expressed as nmol of NADH oxidized/min per mg of SMP.
G. Paradies et al. / Gene 286 (2002) 135–141
137
2.6. Analysis of cardiolipin in SMP Cardiolipin was analyzed by high-pressure liquid chromatography (HPLC), using a Beckman 344 liquid chromatograph. Lipids from beef-heart SMP were extracted with chloroform/methanol by the procedure of Bligh and Dyer (1959). Phospholipids were separated by the HPLC method previously described (Ruggiero et al., 1984) with an Altex ultrasil-Si column (4:6 £ 250 mm). The chromatographic system was programmed for gradient elution using two mobile phases: solvent A, hexane/2-propanol (6:8 v/v) and solvent B, hexane/2-propanol/water (6:8:1.4, v/v/v). The percentage of solvent B in solvent A was increased in 15 min from 0% to 100%. Flow rate was 2 ml/min and detection at 206 nm. 2.7. Preparation of peroxidized cardiolipin Cardiolipin was peroxidized in the presence of Fe 21/ ADP/ascorbic acid as previously described (Paradies et al., 1997a). 2.8. Statistical analysis Results were expressed as mean ^ SE. Statistical significance was determined by the Student’s t-test. 3. Results Cardiolipin is specifically required for optimal functioning of mitochondrial complex I (Fry and Green, 1981). In order to obtain further evidence for the involvement of cardiolipin in the mitochondrial complex I activity, we studied the effect of NAO on the activity of this enzyme complex in submitochondrial particles. NAO has been shown to specifically interact with cardiolipin molecule in the inner mitochondrial membrane (Petit et al., 1992) Fig. 1 shows the UV spectrum of NAO which exhibits an absorp-
Fig. 1. Spectral changes associated with the addition of cardiolipin, phosphatidylcholine or phosphatidylethanolamine to 10-N-nonyl acridine orange. (a) UV-absorption spectrum of 3 mM NAO in 50 mM KPT buffer (pH 7.2) at 30 8C; (b) NAO 1 1:5 mM cardiolipin; (c) NAO 1 1:5 mM phosphatidylcholine; (d) NAO 1 1:5 mM phosphatidylethanolamine.
Fig. 2. NAO inhibition of the complex I activity in SMP and the effect of phospholipids. The complex I activity was measured as described in the Section 2. NAO was added in the assay mixture at concentration of 1.5 mM. cardiolipin, phosphatidylcholine and phosphatidylethanolamine were added at concentration of 3 mM. All values are expressed as mean ^ SE of three independent determinations.
tion maximum at 490 nm. Addition of cardiolipin to a solution of NAO produces a rapid fall in E490 and a new UV spectrum, while the addition of phosphatidylcholine and phosphatidylethanolamine do not modify appreciably the NAO UV spectrum. These spectral changes demonstrate a specific interaction between cardiolipin and NAO. Due to the interaction between NAO and cardiolipin, we tested the effect of NAO on the activity of complex I in submitochondrial particles. The results reported in Fig. 2 show that NAO, at micromolar concentration, strongly inhibited the complex I activity (50% inhibition being achieved at 1.5 mM NAO). The inhibition of complex I by NAO could be largely prevented by the addition of cardiolipin. No prevention was obtained with other phospholipid components of the inner mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine. These results demonstrate that cardiolipin molecules are specifically required for the mitochondrial complex I activity confirming previous results reported by others (Fry and Green, 1981). Thus, alterations in the content and/or in the composition of cardiolipin in the inner mitochondrial membrane may lead to impairment of complex I activity. Oxidative damage of cardiolipin due to ROS attack may represent one way of cardiolipin alteration in the mitochondrial inner membrane. This possibility was explored by studying the effect of ROS, generated by the xanthine/ xanthine oxidase system, on the activity of complex I in submitochondrial particles. The reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide Oz2 2 and
138
G. Paradies et al. / Gene 286 (2002) 135–141
Fig. 3. Production of superoxide anion in bovine heart SMP supplemented with xanthine 1 xanthine oxidase. Superoxide anion production was estimated spectrophotometrically by the SOD-sensitive reduction of acetylated cytochrome c as described in Section 2. The experiment shown is representative of five experiments which gave similar results.
hydrogen peroxide H2O2. Fig. 3 shows the production of superoxide anion in SMP in the presence of X/XO. This production of ROS could be totally blocked by the addition of SOD. The activity of complex I was measured on SMP treated with X/XO, namely under conditions of ROS production. The results reported in Fig. 4 show that exposure of SMP to ROS production resulted in a marked loss of complex I activity (approximately 38% inactivation with 1 h of treatment). Addition of SOD 1 catalase to X/XO treated SMP completely prevented the loss of complex I activity. These
Fig. 4. Oxygen free radical-induced loss of complex I activity in SMP and prevention by SOD 1 catalase. Complex I activity was measured on SMP incubated for 60 min in the absence (control) or presence of xanthine 1 xanthine oxidase as described in Section 2. Where indicated SOD (68 units) and catalase (94 units) were added in the incubation medium before the addition of X/XO. All values are expressed as mean ^ SE of three independent determinations.
results indicate a direct involvement of ROS in this inactivation. The ROS-induced inactivation of complex I could be due to a lower content of cardiolipin which is required for the optimal functioning of this enzyme complex, due to ROS attack on double bonds fatty acids constituents of the cardiolipin molecules. To assess this, the content of cardiolipin was determined in SMP treated for 1 h with X/XO. As shown in Fig. 5 the content of cardiolipin decreased by around 40% in SMP treated with X/XO when compared to the value obtained with untreated SMP. Addition of SOD 1 catalase to X/XO treated SMP, completely prevented the loss of cardiolipin content. It should be noted that there exists a good correlation between the loss of cardiolipin content and the loss of complex I activity. Further evidence for the involvement of cardiolipin in the ROS-induced loss of mitochondrial complex I activity comes from the experiment reported in Fig. 6. Here, it is shown that ROS-induced loss of complex I activity could be largely prevented by exogenous added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipid components of the inner mitochondrial membrane such as phosphatidylcholine or phosphatidylethanolamine, nor by peroxidized cardiolipin. 4. Discussion The mitochondrial electron transport chain has been recognized as major intracellular source of reactive oxygen
Fig. 5. Oxygen free radical-induced loss of cardiolipin content in bovine heart SMP and prevention by SOD 1 catalase. SMP were incubated for 60 min in the absence (control) or presence of X/XO as described in Section 2, and cardiolipin content was determined by the HPLC technique. SOD (68 units) and catalase (94 units) were added in the incubation medium before the addition of X/XO. All values are expressed as mean ^ SE of three independent determinations.
G. Paradies et al. / Gene 286 (2002) 135–141
Fig. 6. Oxygen free radical-induced loss of complex I activity in bovine heart SMP and reactivation by cardiolipin. Complex I activity was measured on SMP incubated for 60 min in the absence (control) or presence of X/XO. Where indicated, cardiolipin, phosphatidylcholine, phosphatidylethanolamine and peroxidized cardiolipin were added to SMP by ethanolic injection at concentration of 3 mM. All values are expressed as mean ^ SE of three independent determinations.
species (Turrens and Boveris, 1980). Under normal physiological conditions, mitochondria contain sufficient levels of antioxidants that prevent ROS generation and oxidative damage. The mitochondrial antioxidant defense mechanism includes both enzymes, such as superoxide dismutase, glutathione peroxidase and glutathione reductase and nonenzymatic substances such as glutathione and a-tocopherol and coenzyme Q. However, under conditions in which excess mitochondrial ROS is produced or when antioxidants are depleted, or both, oxidative damage to mitochondrial can occur. The production of ROS leads to primary reaction and damage in the immediate surrounding of where these reactive oxygen species are produced, by virtue of their high chemical reactivity (Slater, 1984). Therefore, the effects of these reactive oxygen species should be greatest at the level of mitochondrial membrane constituents including the complexes of the respiratory chain (Zhang et al., 1990) and lipid constituents particularly rich in unsaturated fatty acids such as cardiolipin (Paradies et al., 1998, 2000, 2001). Cardiolipin has a particularly important function in mitochondrial bioenergetics in that it interacts with a number of major integral inner membrane proteins including anion carriers and complexes of respiratory chain, even if its precise mechanism of action is still not well understood (Hoch, 1992; Robinson, 1993). Perhaps the best known of these interactions is the requirement of cardiolipin for the cytochrome c oxidase functioning (Vik et al., 1981; Awasthi et al., 1970; Fry et al., 1980; Robinson et al., 1980; Powell et al., 1987; Abramovitch et al., 1990). In this respect, results
139
by this and other laboratories have demonstrated alterations of cytochrome c oxidase activity in mitochondria isolated from animals under different pathophysiological conditions (Paradies et al., 1993a,b, 1994a,b, 1997a,b, 1999). These alterations have been associated to specific changes in the mitochondrial cardiolipin content. It has been reported that cardiolipin is specifically required for electron transfer in complex I of the mitochondrial respiratory chain (Fry and Green, 1981). Consistent with this, it has been found that yeast mutants, deficient in cardiolipin, showed a defect in the mitochondrial respiration due to a prominent deficiency in complex I activity, whereas this activity was restored in a revertant of the mutant that had regained the ability to synthesize cardiolipin (Ohtsuka et al., 1993). In this paper we present further evidence for the cardiolipin involvement in the mitochondrial complex I activity. In fact, we found that 10-N-nonyl-l-acridine, a compound which interacts specifically with cardiolipin in the inner mitochondrial membrane (Petit et al., 1992; Paradies et al., 2001) strongly inhibited the complex I activity in SMP. This inhibition was prevented by cardiolipin, while other phospholipid components such as phosphatidylcholine and phosphatidylethanolamine were ineffective in this respect. These results clearly indicate that cardiolipin is specifically required for the activity of mitochondrial complex I although they do not explain the exact mechanism by which this phospholipid affects the activity of this multisubunit enzyme complex. It is conceivable that alterations in the structure and/or in the content of cardiolipin in the mitochondrial membrane may affect the activity of the complex I. The content of cardiolipin in the inner mitochondrial membrane may change either as a consequence of an alteration of one of the enzymatic steps involved in its biosynthetic process (Schlame and Hostetler, 1997; Hatch, 1996) or as a consequence of peroxidative attack by reactive oxygen species (Paradies et al., 1998, 2000, 2001). In fact, cardiolipin, due to its high content of unsaturated fatty acids (almost 90% represented by linoleic acid), and because of its location in the inner mitochondrial membrane, near to the site of oxygen free radical production, is particularly susceptible to oxidative attack by oxyradicals. Previous works from this laboratory have shown that mitochondrial-mediated ROS generation affects the activity of complex III and IV through peroxidative damage of cardiolipin (Paradies et al., 2000, 2001). The results presented in this study demonstrate that ROS, produced through the use of X/XO system, induce a marked loss of complex I activity in SMP. This loss is completely abolished by SOD and catalase, indicating a direct involvement of ROS in this effect. As mentioned above, cardiolipin is specifically required for the mitochondrial complex I enzyme activity (Fry and Green, 1981). Our results demonstrate that ROS generation leads to a marked loss in the mitochondrial cardiolipin content. This loss of cardiolipin is associated to a parallel loss in the complex I activity. Both
140
G. Paradies et al. / Gene 286 (2002) 135–141
these effects are completely abolished by SOD 1 catalase, which prevent ROS production. In addition, the ROSinduced loss of complex I activity was almost completely restored to the level of untreated SMP by exogenous added cardiolipin, while no restoration was obtained with other major phospholipid constituents of the mitochondrial inner membrane such as phosphatidylcholine and phosphatidylethanolamine nor by peroxidized cardiolipin. Taken together, our results clearly demonstrate that ROS affect the mitochondrial complex I activity through cardiolipin peroxidation, due to oxyradical attack to double bonds of its fatty acid constituents. The impairment of complex I activity and that of complex III and IV previously reported (Paradies et al., 2000, 2001), due to the ROS-induced cardiolipin peroxidation, may increase the electron leak from the electron transport chain, generating more superoxide radical and perpetuating a cycle of oxygen radical-induced damage to mitochondrial membrane constituents. In addition to complexes I, III and IV, cardiolipin serves as co-factor for a number of other mitochondrial membrane proteins involved in mitochondrial bioenergetics, including the ADP/ATP translocator (Hoffmann et al., 1994). Thus, the ROS-induced peroxidative damage to cardiolipin may lead to impairment of mitochondrial energy production by the oxidative phosphorylation. This may have important pathophysiological implications. The basal rate of mitochondrial ROS generation may be altered by different physiological or pathological conditions such as aging (Harman, 1981), apoptosis (Kroemer et al., 1995), alcoholism (Bailey et al., 1999), hyperbaric oxygen toxicity (Turrens, 1997) and chronic degenerative diseases including ischemia/reperfusion (McCord, 1988; Paradies et al., 1999), cancer, Alzheimer’s and chronic inflammation (Wallace, 1992). The pattern of results presented here, together with those previously reported (Paradies et al., 2000, 2001), may prove useful in probing the molecular mechanisms of ROS-induced peroxidative damage to mitochondrial membrane constituents which have been proposed to contribute to the development of these physiopathological conditions and degenerative diseases, and in the development of effective antioxidant strategies.
Acknowledgements This work has been accomplished with funds from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST) and Consiglio Nazionale delle Ricerche.
References Abramovitch, D.A., Marsh, D., Powell, G.L., 1990. Activation of beef-heart cytochrome c oxidase by cardiolipin and analogues of cardiolipin. Biochim. Biophys. Acta 1020, 34–42. Awasthi, Y.C., Chuang, T.F., Keenen, T.W., Crane, F.L., 1970. Association
of cardiolipin and cytochrome oxidase. Biochem. Biophys. Res. Commun. 39, 822–832. Bailey, S.M., Pietsch, E.C., Cunningham, C.C., 1999. Ethanol stimulates the production of reactive oxygen species at mitochondrial complexes I and III. Free Radic. Biol. Med. 27, 891–900. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Boveris, A., Chance, B., 1973. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem. J. 134, 707–716. Boveris, A., 1984. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods Enzymol. 105, 429– 435. Cadenas, E., Boveris, A., Ragan, C.I., Stoppani, A.O.M., 1977. Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch. Biochem. Biophys. 180, 248–257. Chance, B., Sies, H., Boveris, A., 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527–605. Fry, M., Green, D.E., 1981. Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain. J. Biol. Chem. 256, 1874–1880. Fry, M., Blondin, G.A., Green, D.E., 1980. The localization of tightly bound cardiolipin in cytochrome oxidase. J. Biol. Chem. 255, 9967– 9970. Harman, D., 1981. The aging process. Proc. Natl. Acad. Sci. USA 78, 7124–7128. Hatch, G.M., 1996. Regulation of cardiolipin biosynthesis in the heart. Mol. Cell Biochem. 159, 139–148. Hoch, F.L., 1992. Cardiolipins and biomembrane function. Biochim. Biophys. Acta 1113, 71–133. Hoffmann, B., Stockl, A., Schlame, M., Beyer, K., Klingenberg, M., 1994. The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants. J. Biol. Chem. 269, 1940–1944. Kroemer, G., Petit, P., Zamzami, N., Vayssiere, J.L., Mignotte, B., 1995. The biochemistry of programmed cell death. FASEB J. 13, 1277–1287. Lo¨ w, H., Vallin, I., 1963. Succinate-linked diphosphopyridine nucleotide reduction in submitochondrial particles. Biochim. Biophys. Acta 69, 361–374. McCord, J.M., 1988. Free radicals and myocardial ischemia: overview and outlook. Free Radic. Biol. Med. 4, 9–14. Ohtsuka, T., Nishijima, M., Suzuki, K., Akamatsu, Y., 1993. Mitochondrial dysfunction of a cultured Chinese hamster ovary cell mutant deficient in cardiolipin. J. Biol. Chem. 268, 22914–22919. Paradies, G., Ruggiero, F.M., Petrosillo, G., Quagliariello, E., 1993a. Agedependent decrease in the cytochrome c oxidase activity and changes in phospholipid in rat-heart mitochondria. Arch. Gerontol. Geriatr. 16, 263–272. Paradies, G., Ruggiero, F.M., Dinoi, P., Petrosillo, G., Quagliariello, E., 1993b. Decreased cytochrome oxidase activity and changes in phospholipids in heart mitochondria from hypothyroid rats. Arch. Biochem. Biophys. 307, 91–95. Paradies, G., Ruggiero, F.M., Petrosillo, G., Gadaleta, M.N., Quagliariello, E., 1994a. Effect of aging and acetyl-L-carnitine on the activity of cytochrome oxidase and adenine nucleotide. FEBS Lett. 350, 213–215. Paradies, G., Ruggiero, F.M., Petrosillo, G., Quagliariello, E., 1994b. Enhanced cytochrome oxidase activity and modification of lipids in heart mitochondria from hyperthyroid rats. Biochim. Biophys. Acta 1225, 165–170. Paradies, G., Ruggiero, F.M., Petrosillo, G., Quagliariello, E., 1997a. Agedependent decline in the cytochrome c oxidase activity in rat heart mitochondria: role of cardiolipin. FEBS Lett. 406, 136–138. Paradies, G., Petrosillo, G., Ruggiero, F.M., 1997b. Cardiolipin-dependent decrease of cytochrome c oxidase activity in heart mitochondria from hypothyroid rats. Biochim. Biophys. Acta 1319, 5–8. Paradies, G., Ruggiero, F.M., Petrosillo, G., Quagliariello, E., 1998. Perox-
G. Paradies et al. / Gene 286 (2002) 135–141 idative damage to cardiac mitochondria: cytochrome oxidase and cardiolipin alterations. FEBS Lett. 424, 155–158. Paradies, G., Petrosillo, G., Pistolese, M., Di Venosa, N., Serena, D., Ruggiero, F.M., 1999. Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion. Free Radic. Biol. Med. 27, 42–50. Paradies, G., Petrosillo, G., Pistolese, M., Ruggiero, F.M., 2000. The effect of reactive oxygen species generated from mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles. FEBS Lett. 466, 323–326. Paradies, G., Petrosillo, G., Pistolese, M., Ruggiero, F.M., 2001. Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles. Mitochondrion 1, 159. Petit, J.M., Maftah, A., Ratinaud, M.H., Julien, R., 1992. 10-N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria. Eur. J. Biochem. 209, 267– 273. Powell, G.L., Knowles, P.F., Marsh, D., 1987. Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase. Biochemistry 26, 8138–8145. Robinson, N.C., 1993. Functional binding of cardiolipin to cytochrome c oxidase. J. Bioenerg. Biomembr. 25, 153–163.
141
Robinson, N.C., Strey, F., Talbert, L., 1980. Investigation of the essential boundary layer phospholipids of cytochrome c oxidase using Triton X100 delipidation. Biochemistry 19, 3656–3661. Ruggiero, F.M., Landriscina, C., Gnoni, G.V., Quagliariello, E., 1984. Lipid composition of liver mitochondria and microsomes in hyperthyroid rats. Lipids 19, 171–178. Schlame, M., Hostetler, K.Y., 1997. Cardiolipin synthase from mammalian mitochondria. Biochim. Biophys. Acta 1348, 207–213. Slater, T., 1984. Free-radical mechanism in tissue injury. Biochem. J. 222, 1–15. Turrens, J., 1997. Superoxide production by the mitochondrial respiratory chain. Biosci. Rep. 17, 3–8. Turrens, J.F., Boveris, A., 1980. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191, 421–427. Vik, S.B., Georgevich, G., Capaldi, R.A., 1981. Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 78, 1456–1460. Wallace, D.C., 1992. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science 256, 628–632. Zhang, Y., Marcillat, O., Giulivi, C., Ernster, L., Davies, K.J.A., 1990. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J. Biol. Chem. 265, 16330–16336.
