| We developed a detailed model for Mg2+-actin polymerization and analyzed the kinetics of actin polymerization. This model includes two-step nucleation, elongation, and fragmentation-annealing. The size distribution of actin polymer is considered with a cutoff mechanism. We derived a closed set of non-linear coupled differential equations for describing actin polymerization. We show that both fragmentation and annealing are required for fitting the time course of actin polymerization and the dissociation constant for fragmentation-annealing is dependent on the initial G-actin concentration. Dimer and trimer formation are found to be unfavorable compared with the elongation process. However, trimer formation is more favorable than dimer formation. The dissociation constants obtained by fitting the experimental data to the model for dimer formation, trimer formation, and elongation are 5.5 M and 3.5 × 10 −4M, and 0.1 μM respectively. The dissociation constants for fragmentation-annealing are dependent on actin concentrations. This model allows us to evaluate the weight and number average molecular weight and the polydispersity of actin system.; We analyze the effect of latrunculin A on the kinetics of actin assembly and disassembly. The current view of latrunculin A function is that it binds to actin monomers, forming a complex that cannot participate in polymerization (Coué et al., 1987). According to Coué et al., in vitro, latrunculin A forms a 1:1 complex with G-actin and the equilibrium dissociation constant was obtained as 0.2 μM. The present work reports different value for latrunculin A - actin binding. Using light scattering and fluorescence measurements, latrunculin A linearly inhibits actin polymerization rate and completely blocks actin polymerization at 1:2 ratio of actin:latrunculin A. We show that the stoichiometry and the KD for the latrunculin A-actin complex are obtained as 2.1 ± 0.2 and (8.9 ± 13) × 10−18 M2 respectively. Latrunculin A induces actin depolymerization by binding the dissociated actin monomers from the filament ends without binding to F-actin. Latrunculin A does not depolymerize the phalloidin stabilized F-actin. Moreover, latrunculin A induces extremely slow depolymerization of F-actin specimen in vitro. However, actin depolymerization induced by latrunculin A is greatly enhanced by sonication and severing proteins like cofilin and actophorin. Our result suggests that latrunculin A induces rapid disruption in vivo in accordance with severing proteins. |
