HARRIS, VIRGINIA GAIL The Intracellular Targeting of cPLA2. (Under the direction of Scott M. Laster). Tumor necrosis factor-รก (TNF) is an inflammatory cytokine that can induce apoptosis in virus-infected cells, susceptible tumor cells, and cells whose transcriptional or translational processes have been disrupted. The apoptotic pathway that is activated by TNF is dependent on the activity of the enzyme cytosolic phospholipase A2 (cPLA2). This enzyme cleaves arachidonic acid from membrane phospholipid, which in turn causes mitochondrial dysfunction leading to cell death. The goal of this research project was to identify the specific intracellular membrane to which cPLA2 binds during TNF-induced apoptosis. The membrane site for cPLA2 interaction during apoptosis would be a possible target for anti-apoptotic drug development. Three methods were tested in an attempt to identify the membrane location, including; immunofluorescence, biochemical fractionation, and GC/MS. Our results with immunofluorescence suggested that cPLA2 translocates to the nuclear membrane during apoptosis. The identity of the nuclear membrane was confirmed by staining with an antibody to the nuclear pore complex. In addition, cPLA2 staining was noted within the nucleus, perhaps indicating an interaction with chromatin, and small areas of punctate staining were noted in the perinuclear region. Biochemical fractionation indicated that the association of cPLA2 with the nuclear membrane was calcium-dependent since this association could not be stabilized in the absence of calcium. GC/MS, which was used in an attempt to find amputated phospholipids remaining in membranes as a result of cPLA2 activity, instead revealed higher levels of arachidonic acid suggesting that cells may increase the synthesis or repair of arachidonic acid containing membranes following the activation of cPLA2. Finally, our results revealed several novel, lower molecular weight forms of cPLA2 that were associated with nuclei in a calcium-independent fashion. Taken together these results suggest that it will be important to understand the mechanisms controlling the association of cPLA2 with nuclei.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-09012002-190214 |
| Date | 23 September 2002 |
| Creators | Harris, Virginia Gail |
| Contributors | Scott Laster, Amy Grunden, Geraldine Luginbuhl |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-09012002-190214/ |
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