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Identification of Functionally Important Cysteines in the α-Subunit of Transducin by Chemical Cross-Linking Techniques

Journal of Protein Chemistry volume 21, pages 1–8 (2002)Cite this article

Abstract

Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, Tα and Tβγ. N, N′-1, 2-phenylenedimaleimide (o-PDM) and N, N′-1, 4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of Tα with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and ≥200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of Tβγ with both reagents produced a major species of 46-kDa. The combination of intact Tα and o-PDM- or p-PDM-treated Tβγ reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified Tα was incubated with intact Tβγ, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on Tα represent functionally important residues of T.

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REFERENCES

  • Baehr, W., Devlin, M. J., and Applebury, M. L. (1979). J. Biol. Chem. 254, 11669-11677.

    Google Scholar 

  • Baehr, W., Morita, E. A., Swanson, R. J., and Applebury, M. L. (1982). J. Biol. Chem. 257, 6452-6460.

    Google Scholar 

  • Bradford, M. M. (1976). Anal. Biochem. 72, 248-254.

    Google Scholar 

  • Bubis, J. (1995). Biol. Res. 28, 291-299.

    Google Scholar 

  • Bubis, J. (1998). Biol. Res. 31, 59-71.

    Google Scholar 

  • Bubis, J. and Khorana, H. G. (1990). J. Biol. Chem. 265, 12995-12999.

    Google Scholar 

  • Bubis, J. and Ysern, X. (1983). Comp. Biochem. Physiol. 75B, 245-253.

    Google Scholar 

  • Bubis, J., Millan, E. J., and Martinez, R. (1993). Biol. Res. 26, 177-188.

    Google Scholar 

  • Bubis, J., Ortiz, J. O., and Möller, C. (2001). Arch. Biochem. Biophys. 395, 146-157.

    Google Scholar 

  • Bubis, J., Ortiz, J. O., Möller, C., and Millán, E. J. (1994). J. Protein Chem. 13, 473-474.

    Google Scholar 

  • Bubis, J., Ortiz, J. O., Möller, C., and Millán, E. J. (1995). In Methods in Protein Structure Analysis (Atassi, M. Z., and Appella, E., eds.), Plenum Press, New York, pp. 227-250.

    Google Scholar 

  • Dhanasekaran, N., Wessling-Resnick, M., Kelleher, D. J., Johnson, G. L., and Ruoho, A. E. (1988). J. Biol. Chem. 263, 17942-17950.

    Google Scholar 

  • First, E. A., Bubis, J., and Taylor, S. S. (1988). J. Biol. Chem. 263, 5176-5182.

    Google Scholar 

  • Hepler, J. R. and Gilman, A. G. (1992). Trends Biochem. Sci. 17, 383-387.

    Google Scholar 

  • Hingorani, V. N., Tobias, D. T., Henderson, J. T., and Ho, Y.-K. (1988). J. Biol. Chem. 263, 6916-6926.

    Google Scholar 

  • Laemmli, U. K. (1970). Nature 227, 680-685.

    Google Scholar 

  • Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995). Science 270, 954-960.

    Google Scholar 

  • Ortiz, J. O., and Bubis, J. (2001). Arch. Biochem. Biophys. 387, 233-242.

    Google Scholar 

  • Peters, K. and Richards, F. M. (1977). Annu. Rev. Biochem. 46, 523-551.

    Google Scholar 

  • Shichi, H. and Somers, R. L. (1978). J. Biol. Chem. 253, 7040-7046.

    Google Scholar 

  • Towbin, H., Staehlin, T., and Gordon, J. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354.

    Google Scholar 

  • Vaillancourt, R. R., Dhanasekaram, N., Johnson, G. L., and Ruoho, A. E. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 3645-3649.

    Google Scholar 

  • Van Dop, C., Yamanaka, G., Steinberg, F., Sekura, R. D., Manclark, C. R., Stryer, L., and Bourne, H. R. (1984). J. Biol. Chem. 259, 23-26.

    Google Scholar 

  • Wald, G. and Brown, P. (1953-1954). J. Gen. Physiol. 37, 189-200.

    Google Scholar 

  • Wessling-Resnick, M. and Johnson, G. L. (1987a). J. Biol. Chem. 262, 3697-3705.

    Google Scholar 

  • Wessling-Resnick, M. and Johnson, G. L. (1987b). J. Biol. Chem. 262, 12444-12447.

    Google Scholar 

  • Wessling-Resnick, M. and Johnson, G. L. (1989). Biochem. Biophys. Res. Comm. 159, 651-657.

    Google Scholar 

  • West, R. E., Moss, J., Vaughan, M., Liu, T., and Liu, T.-Y. (1985). J. Biol. Chem. 260, 14428-14430.

    Google Scholar 

  • Willardson, B. M., Pou, B., Yoshida, T., and Bitensky, M. W. (1993). J. Biol. Chem. 268, 6371-6382.

    Google Scholar 

  • Yang, C.-S., Skiba, N. P., Mazzoni, M. R., and Hamm, H. E. (1999). J. Biol. Chem. 274, 2379-2385.

    Google Scholar 

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  1. Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela

    Enrique J. Millán & José Bubis

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  1. Enrique J. Millán
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  2. José Bubis
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Correspondence to José Bubis.

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Millán, E.J., Bubis, J. Identification of Functionally Important Cysteines in the α-Subunit of Transducin by Chemical Cross-Linking Techniques. J Protein Chem 21, 1–8 (2002). https://doi.org/10.1023/A:1014174630063

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  • DOI: https://doi.org/10.1023/A:1014174630063

  • Visual process
  • transducin
  • cysteine labeling
  • chemical cross-linking
  • G-protein-coupled signaling