Part I: Characterization of Zn reconstituted Cytochrome B561 Part II: Studies of Catecholamine metabolism in SH-SY5Y and MN9D cells
Hewawitharana, Inoka Samanthi
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Cytochrome b561, a transmembrane heme protein present in neurotransmitter storage vesicles, shuttles electrons from the cytosolic Asc to the intragranullar matrix to regenerate Asc. Although Cyt b561 has been purified, cloned and sequenced from various sources, the physical structure is yet unresolved. Previous studies have shown that one of the hemes in the protein is pH labile under mild alkaline conditions. In the present study, Zn (II) protoporphyrin IX (ZnP) was reconstituted using the altered protein to further study the mechanism of the transmembrane electron transfer reaction of Cyt b561. The ZnP reconstituted protein was found to quench 90% of the fluorescence compared to the same concentration of free ZnP in the solution. The Significance of these findings with respect to the physiological role of Cyt b561 is discussed. In part II, the research has focused on two commonly using CNS dopaminergic models, SH-SY5Y and MN9D cells, which are poorly characterized with respect to their catecholamine metabolism. Differentiated and undifferentiated cells of both cell lines were used to detect the baseline levels and uptake studies. After performing the kinetic experiments for low, medium and high passage cells, we concluded that SH-SY5Y could not be considered as a fully functional CNS dopaminergic model and medium and high passage cells may be characterized as noradrenergic. MN9D cells store high baseline levels of DA but both the differentiated and undifferentiated forms show poor catecholamine uptake characteristics, probably due to the high intracellular DA levels. Therefore, MN9D may be a better CNS dopaminergic model with some deficiencies in DA uptake and release.
Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry