Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 25, 2006
Publication Date: October 3, 2006
Citation: Zukas, A.A., Carter, J.M. 2006. A cofactor limits the pmca conversion reaction [Abstract]. Strategies, advances and trends towards protection of society. p 290. Technical Abstract: Protein Misfolding Cyclic Amplification (PMCA) is an in vitro method of converting cellular prion protein (PrPC) to its abnormal isoform (PrPres). This conformation change is associated with pathogenesis. We use PCMA as a model of in vivo conversion in order to identify cofactors in the conversion reaction. Our version of PCMA uses brain homogenate from healthy hamsters, diluted 1/10 in PBS, 1% Triton X-100, 4mM EDTA, with protease inhibitors, and sonicated repeatedly in the presence of a small amount of a homogenate prepared from infected hamster brain. Five seconds of sonication is alternated with 60 minutes of incubation at 37 ºC. We characterize reaction progress via ELISA and Western blot. Prions are probed with a biotinylated 3F4 antibody streptavidin-AP complex and visualized using BCIP/NBT precipitating dye. When analyzed by Western blot of non-denaturing polyacrylamide gels or native ELISA, the PrPC concentration appears to decrease rapidly within the first 5 seconds of sonication. However, upon analysis by Western blot of denaturing polyacrylamide gels, the concentration of PrPC does not change. This suggests a presence of an intermediate PrPC conformer as a first step in the conversion process of PrPC to PrPres by PMCA. We have also observed that the PCMA reaction is not limited by the supply of PrPC. Conversion slows over time, eventually coming to a stop, even though nearly half the starting amount of PrPC remains. This suggests PCMA requires interaction between PrPC and other cofactors present in brain homogenate. The cofactors appear to be the limiting reagents that are consumed in the conversion process and not recycled. Addition of fresh brain homogenate restores the conversion. These findings provide a new set of tools for studying the cofactor dependent conversion process and understanding disease pathogenesis.