Date of Award
12-22-2011
Document Type
Thesis
Degree Name
Biology, MS
First Advisor
Malathi Srivatsan
Committee Members
Amy Pearce; Anne Grippo
Call Number
LD251 .A566t 2011 K83
Abstract
The fundamental role of enzyme acetylcholinesterase (AChE) is to catalyze the neurotransmitter acetylcholine at cholinergic synapses. In addition to its catalytic activity, AChE appears to play a significant non-catalytic role of neurotropic activity on dorsal root ganglion (DRG) neurons of rat. However the mechanism responsible for AChE's neurotrophic activity is not fully understood. Earlier experiments in Dr. Srivatsan's laboratory demonstrated the binding of fluorophore conjugated AChE to cell surface of DRG neurons. These findings led to my hypothesis that AChE may bind to cell surface protein(s) in DRG neurons, and the resulting protein-protein interaction is responsible for activating downstream signaling and ensuing in AChE neurotrophic activity. To investigate this, my research focused on purifying AChE from fetal bovine serum by affinity chromatography, testing purified AChE for its neurotrophic activity on DRG neurons, isolating the protein(s) that interact with AChE by pull down assay and charactering the same through matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). Results from affinity chromatography method show that AChE was purified successfully from FBS with an average yield of 51.24%. Native gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis of column-purified AChE suggests that AChE retained its catalytic activity during purification steps and was found to be electrophoretically pure respectively. Further, column-purified AChE acted as neurotrophic factor for DRG neurons by significantly enhancing cell survival (p<0.001) and promoting neurite outgrowth. Results from [(2-(4-Hydroxyazobenzene)] Benzoic Acid (HABA) assay indicate that approximately 11 molecules of biotin were coupled to each molecule of AChE suggesting a sufficient biotinylation. Finally, the results from pull down assay demonstrate that AChE appears to interact with an apparent 9 kDa protein in DRG neurons. However, the role of this interaction in AChE's neurotrophic action awaits further studies.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Kunala, Naga Venkata Pavan Kumar, "Mechanism Of Acetylcholinesterase's Neurotrophic Action on DRG Neurons of Rat" (2011). Student Theses and Dissertations. 894.
https://arch.astate.edu/all-etd/894
