Abstract
We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).
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REFERENCES
Ciechanover, A. and Schwatz, A. L. (1998). Proc. Natl. Acad. Sci. USA 95, 2727–2730.
Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982). J. Biol. Chem. 257, 2537–2542.
Copeland, R. A. (2000). Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis, 2nd ed., Wiley-VCH, New York.
Cox, M. J., Shapira, R., and Wilkinson, K. D. (1986). Anal. Biochem. 154, 345–352.
Haas, A. L. and Rose, I. A. (1982). J. Biol. Chem. 257, 10329–10337.
Haas, A. L., Warms, J. V. B., Hershko, A., and Rose, I. A. (1982). J. Biol. Chem. 257, 2543–2548.
Haugland, R. P. (1996). Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, Oregon.
Hershko, A. and Ciechanover, A. (1998). Annu. Rev. Biochem. 67, 425–479.
Jentsch, S. (1992). Annu. Rev. Genet. 26, 179–207.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randal, R. J. (1951). J. Biol. Chem. 193, 265–267.
Macdonald, J. M., LeBlanc, D. A., Haas, A. L., and London, R. E. (1999). Biochem. Pharmacol. 57, 1233–1244.
Patton, E. E., Willems, A. R., and Tyers, M. (1998). Trends Genet. 14, 236–243.
Phillo, J. S. (1994). In Modern Analytical Ultracentrifugation (Schuster, T. M., and Laue, T. M., eds), Birkhauser, Boston.
Piotrowski, J., Beal, R., Hoffman, L., Wilkinson, K. D., Cohen, R. E., and Pickart, C. M. (1997). J. Biol. Chem. 272, 23712–23721.
Prasad, T. and Madura, K. (1998). Anal. Biochem. 260, 135–141.
Rolfe, M., Beer-Romero, P., Glass, S., Eckstein, J., Berdo, I., Theodoras, A., Pagano, M., and Draetta, G. (1995). Proc. Natl. Acad. Sci. USA 92, 3264–3268.
Scheffner, M., Huibregtse, J. M., Vierstra, R. D., and Howley, P. M. (1993). Cell 75, 495–505.
Scheffner, M., Nuber, U., and Huibregtse, J. M. (1995). Nature 373, 81–83.
Tyers, M. and Jorgensen, P. (2000). Curr. Opin. Gen. Dev. 10, 54–64.
Van Holde, K. E. (1985). Physical Biochemistry, Prentice-Hall, Englewood Cliffs, New Jersey.
Vijay-Kumar, S., Bugg, C. E., and Cook, W. J. (1987). J. Mol. Biol. 194, 531–544.
Voges, D., Zwicki, P., and Baumeister, W. (1999). Annu. Rev. Biochem. 68, 1015–1068.
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Wee, K.E., Lai, Z., Auger, K.R. et al. Steady-State Kinetic Analysis of Human Ubiquitin-Activating Enzyme (E1) Using a Fluorescently Labeled Ubiquitin Substrate. J Protein Chem 19, 489–498 (2000). https://doi.org/10.1023/A:1026501515450
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DOI: https://doi.org/10.1023/A:1026501515450