Figure 1.
Biochemical comparison of purified SpCP and MmCP. The conditions were as follows: 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.2 mM ATP, 90 μM CaCl2, and 0.25% glycerol at 25°C. (A) A Coomassie Blue-stained gel showing purification of SpCP. Lane 1, bacteria extract; lane 2, 45–65% ammonium sulfate cut; lane 3, DE52 column; lane 4, Hydroxyapatite column; lane 5, Source 15Q column, pH 7.5; and lane 6, Source 15Q column, pH 6.0. Molecular weights are indicated on the left. (B and C) Critical concentration for assembly of rabbit skeletal muscle actin. (B) Dependence of actin polymer concentration on total actin concentration in the (•) absence or presence of either (□) 15 nM MmCP (○) 150 nM SpCP. Actin (5% pyrene labeled) was assembled for 16 h. The polymer concentration was measured from the pyrene fluorescence, plotted versus actin concentration, and fit by linear regression. The Cc values were 0.1 μM for actin alone, 0.95 μM with 15 nM MmCP, and 0.90 M with 150 μ nM SpCP. (C) Dependence of the polymer concentration of 1 μM actin on the concentration of (□) MmCP or (○) SpCP. Actin filaments (5 μM) (10% pyrene labeled) were diluted to 1 μM in the presence of a range of CP concentrations. After 16 h, the pyrene fluorescence was measured, and the actin polymer concentration was plotted versus the log of CP concentration. (D–F) Direct visualization of the effect of capping protein on growing actin filaments by time-lapse evanescent wave fluorescence microscopy. Mg-ATP actin (1.0 μM) with 0.25 or 0.65 μM Oregon green (OG) 488-labeled Mg-ATP actin. Black triangles indicate time when the second solution replaced the initial solution. Left, kymograph of the length (y-axis) of a representative filament versus time (x-axis; 1200 s). Right, lifetime plots for eight filaments of the growth of barbed and pointed ends versus time. An initial solution that contained 1.0 μM actin with 0.25 μM OG-actin was followed by a second solution that contained 1.0 μM actin with 0.65 μM OG-actin (D) alone, or with either (E) 10 nM MmCP or (F) 250 nM SpCP. (G and H) Barbed end addition of monomer to preassembled actin filaments in the presence of capping protein. (G) Time course of the assembly of 1 μM actin (5% pyrene labeled) saturated with 5 μM profilin upon addition to 400 nM actin filaments (thick curve) alone, or in the presence of either (•) 2.5, (▪) 5 and (□) 10 nM MmCP or (○) 10, (□) 35 and (□) 100 nM SpCP. (H) Dependence of the initial rate of barbed end assembly on the concentration of (□) MmCP or (○) SpCP. Curve fits of the plotted data (see Materials and Methods) revealed dissociation equilibrium constants of 0.8 nM for MmCP and 16 nM for SpCP. (I and J) Time course of the depolymerization of 5 μM actin filaments (70% pyrene labeled) after dilution to 0.1 μM in the (thick curve) absence or presence of (□) 1 nM MmCP, (▪) 5 nM MmCP, (○) 10 nM SpCP, and (•) 250 nM SpCP. (J) Dependence of the rate of depolymerization on the concentration of (□) MmCP or (○) SpCP. The data from 300 to 1000 s of each curve was fit with single exponentials, and the depolymerization rates were expressed as a fraction of the rate of actin alone. (K and L) Time course of the spontaneous assembly of 4 μM Mg-ATP actin (5% pyrene labeled) in the absence (thick curve) or presence of either a range of concentrations of MmCP (•) 5 nM, (▪) 25 nM, and (□) 100 nM, or a range of concentrations of SpCP, (□) 5 nM, (○) 25 nM, and (□) 100 nM. (L) Biphasic dependence of the concentration of apparent ends (nanomolar) on the concentration of SpCP calculated from the rate of polymerization at the time where 25% (1 μM) of the actin was polymerized. (M) Effects of capping protein on actin filament annealing. Merged micrographs of red and green fluorescence are shown. Equal concentrations (0.25 μM) of red (rhodamine-phalloidin)- and green (Alexa green-phalloidin)–labeled actin filaments were sheared through a 26-gauge needle in the absence or presence of the indicated concentrations of CP and allowed to anneal for 60 min before dilution and absorption to poly-l-lysine–coated coverslips. Bar, 5 μm.