The integrity and dynamics of the actin cytoskeleton is essential for the morphology and motility of neuronal cells both in developing and adult nervous system. Oxidative modification of actin both as monomers and in filaments alters the kinetics of actin filament dynamics contributing to neurodegeneration and aging. Our objective focused on changes of the kinetic parameters of oxidized actin as opposed to naive actin and its resulting effects on actin filament dynamics. We first validated Surface Plasmon Resonance (SPR) technology for our objective by studying interaction of naive actin with a monoclonal anti-actin antibody and DNase I. For interactions of actin monomers under polymerizing conditions yet below the critical concentration of actin, we determined real time rate constants for actin nucleation steps for the first time. The association rate constant (ka) for the first monomer was 7.5x10³ μM⁻¹sec⁻¹ and the dissociation rate constant (ka) for first monomer was 0.256 sec⁻¹. Whereas Ka for second monomer remained unchanged (6.66x10³ μM⁻¹sec⁻¹), kd for second monomer was significantly decreased (1.82x10⁻³ sec⁻¹). We also found that affinity of oxidized actin to unoxidized actin is significantly (100 fold) reduced. Our findings demonstrate that SPR allows measurement of (kinetic parameters) for unmodified actin monomers.
Thesis (M.S.) University of Alaska Fairbanks, 2007
Table of Contents
1. Physiology of actin dynamics -- 2. Oxidative stress and oxidative modifications -- 3. Oxidative modifications of cellular actin cytoskeleton -- 4. Methods -- 5. Interaction of actin with monoclonal anti-actin antibody and DNase I -- 6. Interaction of actin with ATP -- 7. Actin-actin interaction -- 8. Overall conclusions -- 9. Future directions -- 10. References.
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