Abstract
We have previously shown that mitochondrial membrane potential (δψ) drop promoted by prooxidants and Ca2+ can be reversed but not sustained by ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or δψ elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after δψ was collapsed. Total δψ recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during δψ drop continues even after δψ is already eliminated. Titration with 5,5′-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of δΩ drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.
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References
Al-Nasser, I., and Crompton, M. (1986).Biochem. J. 239, 19–29.
Bernardes, C. F., Meyer-Fernandes, J. R., Basseres, D. S., Castilho, R. F., and Vercesi, A. E. (1994).Biochim. Biophys. Acta 1188, 93–100.
Bernardi, P., Broekemeier, K., and Pfeiffer, D. (1994).J. Bioenerg. Biomembr. 26, 509–517.
Castilho, R. F., Kowaltowski, A. J., Meinicke, A. R., Bechara, E. J. H., and Vercesi, A. E. (1995a).Free Radical Biol. Med. 18, 479–486.
Castilho, R. F., Kowaltowski, A. J., Meinicke, A. R., and Vercesi, A. E. (1995b).Free Radical Biol. Med. 18, 55–59.
Crompton, M. (1990). InCalcium and the Heart (Langer, G. A., ed.), Raven, New York, pp. 167–198.
Crompton, M., Costi, A., and Hayat, L. (1987).Biochem. J. 245, 915–918.
Fagian, M. M., Pereira-da-Silva, L., Martins, I. S., and Vercesi, A. E. (1990).J. Biol. Chem. 265, 19955–19960.
Gunter, T. E., and Pfeiffer, D. R. (1990).Am. J. Physiol. 258, C755-C786.
Gunter, T. E., Gunter, K. K., Sheu, S. -S., and Gavin, C. E. (1994).Am. J. Physiol. 267, C313-C339.
Harbury, P. B., Zhang, T., Kim, P. S. and Alber, T. (1993).Science 262, 1401–1407.
Igbavboa, U., Zwizinski, C. W., Pfeiffer, D. R. (1989).Biochem. Biophys. Res. Commun. 161, 619–625.
Imberti, R., Nieminen, A. L., Herman, B., Lemasters, J. J. (1992).Res. Commun. Chem. Pathol. Pharmacol. 78, 27–38.
Jensen, B. D., Gunter, K. K., and Gunter, T. E. (1986).Arch. Biochem. Biophys. 248, 305–323.
Kamo, N., Muratsugu, M., Hongoh, R., and Kobatake, J. (1979).J. Membr. Biol. 49, 105–121.
Klingenberg, M. (1989).Arch. Biochem. Biophys. 270, 1–14.
Kowaltowski, A. J., Castilho, R. F., and Vercesi, A. E. (1995).Am. J. Physiol. 269, C141–147.
Kowaltowski, A. J., Castilho, R. F., Vercesi, A. E. (1996a).FEBS Lett. 378, 150–152.
Kowaltowski, A. J., Castilho, R. F., Grijalba, M. T., Bechara, E. J. H., Vercesi, A. E. (1996b).J. Biol. Chem. 271, 2929–2934.
Laemmli, U. K. (1970).Nature 227, 680–685.
Maddaiah, V. T., and Kumbar, U. (1993).J. Bioenerg. Biomembr. 25, 419–427.
Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y., and Terada, H. (1994).Biochemistry 33, 9530–9536.
McEnery, M. W., Snowman, A. M., Trifiletti, R. R., and Snyder, S. H. (1992).Proc. Natl. Acad. Sci. USA 89, 3170–3174.
Muratsugu, M., Kamo, N., Kurihara, K., and Kobatake, J. (1977).Biochim. Biophys. Acta 464, 613–619.
Nazareth, W., Nasser, Y., and Crompton, M. (1991).J. Mol. Cell. Cardiol. 23, 1351–1354.
Novgorodov, S. A., Gudz, T. I., Brierley, G. P., and Pfeiffer, D. R. (1994).Arch. Biochem. Biophys. 311, 219–228.
Pastorino, J. G., Snyder, J. W., Serroni, A., Hoek, J. B., and Farber, J. L. (1993).J. Biol. Chem. 268, 13791–13798.
Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994).J. Biol. Chem. 269, 16638–16642.
Scherer, B., and Klingenberg, M. (1974).Biochemistry 13, 161–169.
Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7.
Vercesi, A. E. (1984).Arch. Biochem. Biophys. 231, 86–91.
Vercesi, A. E. (1993).Brazilian J. Med. Biol. Res. 26, 441–457.
Vercesi, A. E., Castilho, R. F., Meinicke, A. R., Valle, V. G. R., Hermes-Lima, M., and Bechara, E. J. H. (1994).Biochim. Biophys. Acta 1188, 86–92.
Vignais, P. V., and Vignais, P. M. (1972).FEBS Lett 26, 27–31.
Zoratti, M., and SzabÒ, I. (1995).Biochim. Biophys. Acta 1241, 139–176.
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Castilho, R.F., Kowaltowski, A.J. & Vercesi, A.E. The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking. J Bioenerg Biomembr 28, 523–529 (1996). https://doi.org/10.1007/BF02110442
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DOI: https://doi.org/10.1007/BF02110442