دورية
A Monomer-Dimer Equilibrium of a Cellular Prion Protein (PrPC) Not Observed with Recombinant PrP*
| العنوان: | A Monomer-Dimer Equilibrium of a Cellular Prion Protein (PrPC) Not Observed with Recombinant PrP* |
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| المؤلفون: | Meyer, Rudolf K., Lustig, Ariel, Oesch, Bruno, Fatzer, Rosmarie, Zurbriggen, Andreas, Vandevelde, Marc |
| المصدر: | Journal of Biological Chemistry; December 2000, Vol. 275 Issue: 48 p38081-38087, 7p |
| مستخلص: | Both the purified normal (protease-sensitive) isoform of the prion protein (PrPC) (Pergami, P., Jaffe, H., and Safar, J. (1996) Anal. Biochem.236, 63–73) and recombinant prion protein (PrP) have been found to be in monomeric form (Mehlhorn, I., Groth, D., Stockel, J., Moffat, B., Reilly, D., Yansura, D., Willet, W. S., Baldwin, M., Fletterick, R., Cohen, F. E., Vandlen, R., Henner, D., and Prusiner, S. B. (1996)Biochemistry35, 5528–5537; and this paper), and therefore PrPC-PrPCinteractions were previously unknown. In this report we confirm recombinant PrP to be a monomer by analytical ultracentrifugation. However, by three lines of evidence (enzyme-linked immunosorbent assay (ELISA), cross-linking experiments, and size exclusion chromatography) we could also demonstrate that, under native conditions, at least part of the native bovine PrPCexists as a monomer-dimer equilibrium. A bovine PrPC-specific immuno-sandwich ELISA was developed and calibrated with recombinant PrP (Meyer, R. K., Oesch, B., Fatzer, R., Zurbriggen, A., and Vandevelde, M. (1999) J. Virol.73, 9386–9392). By this ELISA we identified a distinct PrPCfraction and partially purified this protein. When serial dilutions of brain homogenate or partially purified PrPCwere measured, using the peptide antibody C15S, a nonlinear dose-response curve was obtained. This nonlinearity was shown not to be due to an artifact of the procedure but to a monomer-dimer equilibrium of PrPCwith preferential binding of the antibody to the dimer. From the curvature we could deduce the association constant (3.9 × 108M−1at 37 °C). Accordingly, ΔG° of the reaction was calculated (−48.6 kJ M−1), and ΔH° (9.5 kJ M−1) as well as ΔS° (0.2 kJ K−1M−1) were extrapolated from the van't Hoff plot. When serial dilutions of monomeric recombinant PrP were tested, only a straight line was obtained, supporting our hypothesis. Additional evidence of dimer formation was revealed by Western blotting of partially purified PrPCcross-linked by the homobifunctional cross-linker BS3. Finally, size exclusion chromatography of partially purified PrPCfractions revealed an additional shoulder not observed with recombinant PrP. The difference in respect of dimer formation between native PrPCand recombinant PrP could be explained by the lack of glycosylation of the latter. |
| قاعدة البيانات: | Supplemental Index |
| تدمد: | 00219258 1083351X |
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| DOI: | 10.1074/jbc.M007114200 |