NR AOXE
AU Mueller,H.; Strom,A.; Stuke,A.W.
TI PrP Isoforms analysed by Immobilised Metal Affinity Chromatography (IMAC)
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-60
PT Konferenz-Poster
AB The host PrPc and the pathogenic PrPsc can be distinguished by deviating Cu2+-affinities. After subcellular fractionation of un- and scrapie-infected whole mouse brain and BSE-infected bovine stem brain all membrane fractions were solubilised with n-octyl-D-glucopyranoside and the PrP isoforms were not only enriched but also separated by IMAC. Comparing the total PrP contents it became obvious that after BSE infection the PrP distribution within the subcellular fractions differed. The PrP concentration was increased 2.5 times. The octarepeats are responsible for the Cu2+-binding of PrPc and denatured PrPsc which were found only in the eluate. In contrast, PrPres as well as PrPsc are distinguished by an inability to be retained by a Cu2+-resin. The differences in retention presumably result from the conformational accessibility of the N-terminus of PrPsc. Whereas PrPc was characterised only by a strong signal for the di-glycosylated form, always three strong PrPsc-signals were observed. Before Cu2+-releasing by EDTA different imidazole concentrations were used to compete for Cu2+-binding and to influence the total protein distribution with respect to flowthrough/wash fraction or eluate. By this means it appeared to be possible to optimise the enrichment of PrPc and denatured PrPsc in the eluate, or native PrPsc and PrPres in the retained fraction. In scrapie-infected specimen 2.2 times and in BSE-infected specimen 8.4 times more PrPsc than PrPc was detectable. Thus, after TSE infection the majority of the available PrP is supposed to be the infectious isoform. After IMAC of scrapie-infected murine brain the protease sensitivity of PrPsc was decreased e.g. by Cu2+ changing the N-terminal conformation. In contrast, in BSE-infected bovine brain only low concentrations of PrPres even without a band displacement were observed. This might reflect a greater protease susceptibility of particular PrPsc conformers rather than indicating low concentrations of infectious prions.
AD H. Mueller, A. Strom, A.W. Stuke, German Primate Centre (DPZ), Germany
SP englisch
PO Deutschland