Linked Life Data

10967124

Source:http://linkedlifedata.com/resource/pubmed/id/10967124

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
48
pubmed:dateCreated
2000-12-18
pubmed:abstractText
Both the purified normal (protease-sensitive) isoform of the prion protein (PrP(C)) (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 PrP(C)-PrP(C) 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 PrP(C) exists as a monomer-dimer equilibrium. A bovine PrP(C)-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 PrP(C) fraction and partially purified this protein. When serial dilutions of brain homogenate or partially purified PrP(C) 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 PrP(C) with preferential binding of the antibody to the dimer. From the curvature we could deduce the association constant (3.9 x 10(8) M(-1) at 37 degrees C). Accordingly, DeltaG degrees of the reaction was calculated (-48.6 kJ M(-1)), and DeltaH degrees (9.5 kJ M(-1)) as well as DeltaS degrees (0.2 kJ K(-1) M(-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 PrP(C) cross-linked by the homobifunctional cross-linker BS(3). Finally, size exclusion chromatography of partially purified PrP(C) fractions revealed an additional shoulder not observed with recombinant PrP. The difference in respect of dimer formation between native PrP(C) and recombinant PrP could be explained by the lack of glycosylation of the latter.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
275
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
38081-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
A monomer-dimer equilibrium of a cellular prion protein (PrPC) not observed with recombinant PrP.
pubmed:affiliation
TSE Reference Center, Institute of Animal Neurology, University of Bern, Bremgartenstrasse 109a, CH-3012 Bern, Switzerland. rudolf.meyer@itn.unibe.ch
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't