Purification and Identification of Ubiquitin Binding Proteins from Erythrocytes of Patients with Dementia

Objective：The continuous synthesis and degradation of proteins in the cell are essential for the maintenance of cellular homeostasis. Intracellular protein degradation largely occurs in the lysosome and cytoplasm. The protein degradation in the cytoplasm (ubiquitin mediated protein degradation) is distinct from the well studied lysosomal protein degradation (nonselective protein degradation) and require energy (ATP), ubiquitin and ubiquitin conjugating enzymes such as E1, E2 and E3. Dementia caused by the deposition of abnormal proteins in brain cells followed by brain cells damage are not fully understood. To better understand the possible mechanism of dementia, we attempted to purify ubiquitin conjugating enzymes (such as E1 and E2 proteins) from the blood of normal persons and patients with dementia and tested their electrophoretic mob)ility on SDS-polyacrylamide gel electrophoresis. Methods：The E1 and E2 enzymes of the red blood cell lysate fraction from the normal person and the patients with dementia were purified from ammonium sulfate precipitatant of DEAEcellulose eluate fraction. Following ubiquitin-sepharose column chromatography, the E1 enzyme of the normal and the patients with dementia group showed homogeneous form and various kinds of E2 isoforms were identified by the SDS-polyacrylamide gel electrophoresis. Results：The E1 and E2 enzymes showed no difference on electrophoretic mobility, but the E2 isozyme containing fraction was observed to great difference between the two groups. The 44 kDa protein of E2 isozyme containing fraction was significantly increased in alcoholic dementia and clearly increased in patients with Alzheimer's disease. In addition, another 11 kDa protein was significantly increased in the patients with Alzheimer's disease, but 11 kDa protein of alcoholic dementia was similar to that of the normal person. The 44 kDa and 11 kDa proteins showed a reverse relationship between alcoholic dementia and the patients with Alzheimer's disease. These proteins seems to be different molecules from the well known studied β-amyloid, presenilin, tau protein and apolipoprotein E (Apo E). Conclusions：These results might be useful for the elucidation of dementia and the identification of these proteins are now in progress.ope