Optimizing Conditions For The Determination of METH and Parkin's Effect on 26S Proteasome Dissociation In Vivo
Muniga, Ellen T.
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The dopaminergic terminals within the brains of many methamphetamine (METH) abusers and Parkinson's disease patients have been found to contain protein aggregates and inclusions containing ubiquitin and other proteins involved in the ubiquitin proteasome system (UPS). The formation of these aggregates could be a result of 26S proteasome dysfunction. 26S's role in the UPS is to degrade defective proteins and short-lived regulatory proteins that have been tagged with ubiquitin by parkin, an E3-ligase operating within the UPS. Moszczynska and Yamamoto found that METH reduces the activity of 26S and oxidatively damages parkin. However, it is unknown if 26S activity decreases due to METH damaging parkin, or from METH causing 26S to dissociate. An experiment utilizing the techniques of native gel electrophoresis and immunoblotting has been proposed to determine if METH promotes the dissociation of 26S and if parkin levels affect 26S dissociation. The goal of this project was to optimize the conditions for this experiment, focusing mainly on the conditions needed for 26S to remain associated during tissue homogenization, native gel electrophoresis and immunoblotting. Once the conditions for 26S association have been established the in-gel levels and activity of 26S in striatal synaptosomes from the brains of wild-type and parkin-overexpressing rats treated with or without methamphetamine can be measured. It was found that ATP, DTT, and MgCl2 are needed in the sample homogenization buffer, as well as the anode and cathode buffers during electrophoresis in order for the assembly of 26S to occur.