AGGREGATES OF SCRAPIE-ASSOCIATED PRION PROTEIN INDUCE THE CELL-FREE CONVERSION OF PROTEASE-SENSITIVE PRION PROTEIN TO THE PROTEASE-RESISTANT STATE

Introduction: Scrapie infection instigates the in vivo conversion of n ormal, protease-sensitive prion protein (PrPC) into a protease-resista nt form (PrPSc) by an unknown mechanism. In vitro studies have indicat ed that PrPSc can induce this conversion, consistent with proposals th at PrPSc itself might be the infectious scrapie agent. Using this cell -free model of the PrPC to PrPSc conversion, we have studied the depen dence of conversion on reactant concentration, and the properties of t he PrPSc-derived species that has converting activity. Results: The ce ll-free conversion of S-35 PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (a bove an apparent minimum threshold concentration) and S-35 PrPC. Analy sis of the physical size of the converting activity indicated that det ectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrPSc and enhanced the converting activity the active species were heterogeneou s in size, hut larger than either effectively solubilized PrP or molec ular weight: standards of similar to 2000 kDa. Conclusions: The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. T hese results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.