title: Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy.
abstract: We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-P(i)-actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 microM almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP.
authors.affiliation: Department of Molecular Biology, Yale University, New Haven, CT 06520-8103, USA.
authors.forename: Ikuko
authors.initials: I
authors.lastname: Fujiwara
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Vavylonis
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pollard
mesh_terms: D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010710:Phosphates / Q000378:metabolism*; D011817:Rabbits; D013816:Thermodynamics
chemical_list: C042634:ADP-G-actin; D000199:Actins; D010710:Phosphates; D000244:Adenosine Diphosphate; D008274:Magnesium