The kinetics of cytochalasin D binding to monomeric actin

It has been shown previously, using G-actin labeled with N-idioacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine, that Mg²⁺ induces a conformational change in monomeric G-actin as a consequence of binding to a tight divalent cation binding site. Using the same fluorescent probe, we show that, subsequent to the Mg²⁺-induced conformational change, cytochalasin D induces a fluorescence decrease. The data are consistent with a mechanism which proposes that, after Mg²⁺ binding, cytochalasin D binds and induces a second conformational change which results in overall tight binding of the cytochalasin. The initial binding of cytochalasin D to monomeric actin labeled with the fluorescent probe was found to be 200 μM, and the forward and reverse rate constants for the subsequent conformational change were 350 s^-1 and 8 s^-1, respectively, with an overall dissociation constant to the Mg²⁺-induced form of 4.6 μM. The conformational change does not occur in monomeric actin in the presence of Ca²⁺ rather than Mg²⁺, but Ca²⁺ competes with Mg²⁺ for the tight binding site on the G-actin molecule. Direct binding studies show that actin which has not been labeled with the fluorophore binds cytochalasin D more tightly. The conformational change induced by Mg²⁺ and cytochalasin D precedes the formation of an actin dimer.