Molecular Properties of Purified Human Uncoupling Protein 2 Refolded from Bacterial Inclusion Bodies

Journal of bioenergetics - Tập 35 - Trang 409-418 - 2003
Mika B. Jekabsons1,2, Karim S. Echtay1, Ignacio Arechaga1, Martin D. Brand1
1Medical Research Council, Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Cambridge, United Kingdom
2Buck Institute for Age Research, Novato

Tóm tắt

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (K d) of 0.3–0.5 and 23–92 μM. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a K d of greater than 100 μM, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-14C] ATP and [8-14C] ADP with K ds of 4–5 and 16–18 μM, respectively. Mg2+ (2 mM) reduced the apparent ATP affinity to 53 μM, an effect entirely explained by chelation of ATP; with Mg2+, K d using calculated free ATP was 3 μM. A combination of gel filtration, Cu2+-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg2+-free nucleotides: the K d for ATP is about 3–5 μM and for Mant-ATP it is about 10 times lower; and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form.

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Tài liệu tham khảo

Arsenijevic, D., Onuma, H., Pecqueur, C., Raimbault, S., Manning, B. S., Miroux, B., Couplan, E., Alves-Guerra, M. C., Goubern, M., Surwit, R., Bouillaud, F., Richard, D., Collins, S., and Ricquier, D. (2000). Nat. Genet. 26, 435–439.

Couplan, E., Del Mar Gonzalez-Barroso, M., Alves-Guerra, M. C., Ricquier, D., Goubern, M., and Bouillaud, F. (2002). J. Biol. Chem. 277, 26268–26275.

Echtay, K. S., Bienengraeber, M., and Klingenberg, M. (1997). Biochemistry 36, 8253–8260.

Echtay, K. S., Roussel, D., St-Pierre, J., Jekabsons, M. B., Cadenas, S., Stuart, J. A., Harper, J. A., Roebuck, S. J., Morrison, A., Pickering, S., Clapham, J. C., and Brand, M. D. (2002). Nature 415, 96–99.

Echtay, K. S., Winkler, E., Bienengraeber, M., and Klingenberg, M. (2000). Biochemistry 39, 3311–3317.

Gong, D. W., Monemdjou, S., Gavrilova, O., Leon, L. R., Marcus-Samuels, B., Chou, C. J., Everett, C., Kozak, L. P., Li, C., Deng, C., Harper, M. E., and Reitman, M. L. (2000). J. Biol. Chem. 275, 16251–16257.

Headrick, J. P., McKirdy, J. C., and Willis, R. J. (1998). Am. J. Physiol. 275, H917–H929.

Huang, S. G., and Klingenberg, M. (1995). Biochemistry 34, 349–360.

Jekabsons, M. B., Echtay, K. S., and Brand, M. D. (2002). Biochem. J. 366, 565–571.

Klingenberg, M. (1988). Biochemistry 27, 781–791.

Klingenberg, M., and Appel, M. (1989). Eur. J. Biochem. 180, 123–131.

Klingenberg, M., Echtay, K. S., Bienengraeber, M., Winkler, E., and Huang, S. G. (1999). Int. J. Obes. Relat. Metab. Disord. (Suppl. 6), S24–S29.

Klingenberg, M., and Huang, S. G. (1999). Biochim. Biophys. Acta 1415, 271–296.

Klingenberg, M., Winkler, E., and Huang, S. G. (1995). Methods Enzymol. 260, 369–389.

Kobashi, K. (1968). Biochim. Biophys. Acta 158, 239–245.

Krauss, S., Zhang, C. Y., and Lowell, B. B. (2002), Proc. Natl. Acad. Sci. U.S.A. 99, 118–122.

Lawson, J. W., and Veech, R. L. (1979). J. Biol. Chem. 254, 6528–6537.

Lee, S. C., Hamilton, J. S., Trammell, T., Horwitz, B. A., and Pappone, P. A. (1994). Am. J. Physiol. 267, C349–C356.

Lin, C. S., Hackenberg, H., and Klingenberg, M. (1980). FEBS Lett. 113, 304–306.

Lin, C. S., and Klingenberg, M. (1980). FEBS Lett. 113, 299–303.

Lin, C. S., and Klingenberg, M. (1982). Biochemistry 21, 2950–2956.

Locke, R. M., Rial, E., and Nicholls, D. G. (1982). Eur. J. Biochem. 129, 381–387.

Moller, J. V., and le Maire, M. (1993). J. Biol. Chem. 268, 18659–18672.

Palmisano, A., Zara, V., Honlinger, A., Vozza, A., Dekker, P. J., Pfanner, N., and Palmieri, F. (1998). Biochem. J. 333, 151–158.

Pecqueur, C., Alves-Guerra, M. C., Gelly, C., Levi-Meyrueis, C., Couplan, E., Collins, S., Ricquier, D., Bouillaud, F., and Miroux, B. (2001). J. Biol. Chem. 276, 8705–8712.

Portman, M. A., Xiao, Y., Song, Y., and Ning, X. H. (1997). Am. J. Physiol. 273, H1977–H1983.

Romani, A. M., and Scarpa, A. (2000). Front. Biosci. 5, D720–D734.

Ronner, P., Naumann, C. M., and Friel, E. (2001). Diabetes 50, 291–300.

Schoroers, A., Burkovski, A., Wohlrab, H., and Kramer, R. (1998). J. Biol. Chem. 273, 14269–14276.

Schwenke, W. D., Soboll, S., Seitz, H. J., and Sies, H. (1981). Biochem. J. 200, 405–408.

Stuart, J. A., Harper, J. A., Brindle, K. M., Jekabsons, M. B., and Brand, M. D. (2001). J. Biol. Chem. 276, 18633–18639.

Zhang, C. Y., Baffy, G., Perret, P., Krauss, S., Peroni, O., Grujic, D., Hagen, T., Vidal-Puig, A. J., Boss, O., Kim, Y. B., Zheng, X. X., Wheeler, M. B., Shulman, G. I., Chan, C. B., and Lowell, B. B. (2001). Cell 105, 745–755.

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Thông tin xuất bản

Journal of bioenergetics

Tập 35

409-418

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