Figure 6

Dynacortin promotes assembly of actin in low-salt conditions in vitro. A. Actin (10 μM) was induced to polymerize under G-buffer conditions. Polymerization was monitored as an increase in fluorescence intensity over time by exciting at 365 nm and collecting emission at 385 nm. The experiment was repeated at least three times at each dynacortin concentration with the same results. Curve 1, 10 μM G-actin alone; curve 2, 10 μM G-actin + 1.3 μM Dyn₂; curve 3, + 2.5 μM Dyn₂; curve 4, + 4 μM Dyn₂; curve 5, + 5 μM Dyn₂; curve 6, + 7.5 μM Dyn₂. B. Observed rates for actin assembly induced by varying concentrations of dynacortin. The slope of the polymerization curve was determined by fitting polymerization data to Equation 3. The slope of the line-fitting the rising part of the curve gives a value of k ⁺/K _M = 0.0014 μM^-1s^-1 and slope of the line-fitting the plateau part of the curve yields a value of k ⁺ = 0.0042 ± 0.0009 s^-1. The calculated K _M is 3 μM. C. Dynacortin binds to G-actin. Varying concentrations of Dyn₂ and 10-μM skeletal muscle actin were mixed in G-buffer, ultracentrifuged and the pellets were analyzed by SDS-PAGE. Concentrations are indicated on gel. D. Dynacortin induced the formation of actin bundles under G-buffer condition. Electron micrograph of grid spotted with polymerization reaction mixture of 3-μM pyrene-labeled actin and 3-μM Dyn₂ 1 000 s after assembly was initiated. Scale bar, 200 nm. E. Dynacortin decreased slightly the lag time of actin assembly under F-buffer conditions. A total of 2.5 μM actin was present in each sample. Curve 1, 2.5 μM actin alone; curve 2, 1.3 μM Dyn₂; curve 3, 5 μM Dyn₂; curve 4, 7.5 μM Dyn₂. F. Dynacortin increased the apparent nucleation rate nearly twofold in a concentration-dependent manner.