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J. Biol. Chem., Vol. 275, Issue 48, 38081-38087, December 1, 2000
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From the 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)
Biochemistry 35, 5528-5537; and this paper), and therefore
PrPC-PrPC interactions 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 PrPC exists
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
PrPC fraction and partially purified this protein. When
serial dilutions of brain homogenate or partially purified
PrPC were 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 PrPC with preferential binding of the
antibody to the dimer. From the curvature we could deduce the
association constant (3.9 × 108
M
A Monomer-Dimer Equilibrium of a Cellular Prion Protein
(PrPC) Not Observed with Recombinant PrP*
§,
,,, and
TSE Reference Center, Institute of Animal
Neurology, University of Bern, Bremgartenstrasse 109a,
CH-3012 Bern, ¶ Biocenter, University of Basel,
Klingelbergstrasse 70, 4056 Basel, and Prionics Ltd.,
University of Zürich, Wintherthurerstrasse 190,
CH-8057 Zürich, Switzerland
1 at 37 °C). Accordingly, 
G° of the
reaction was calculated (
48.6 kJ M1), and
H° (9.5 kJ M1) as well as S° (0.2 kJ K1 M1) 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 PrPC
cross-linked by the homobifunctional cross-linker BS3.
Finally, size exclusion chromatography of partially purified PrPC fractions revealed an additional shoulder not observed
with recombinant PrP. The difference in respect of dimer formation
between native PrPC and recombinant PrP could be explained
by the lack of glycosylation of the latter.
*
This work was supported by a grant from the Swiss Federal
Veterinary Office and by the Swiss Federal Office for Education and
Science (Fair5-CT97-3311).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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