The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin

The stoichiometric actin – DNase-I complex was used to study the actin – nucleotide and actin – divalentcation interactions and its ATPase activity in the presence of MgCl2 and cytochalasin D. Treatment of actin – DNase-I complex with 1 mM EDTA results in almost complete restoration of its otherwise inhibited DNase I activity, although the complex does not dissociate, as verified by size-exclusion chromatography. This effect is due to a loss of actin-bound nucleotide but is prevented by the presence of 0.1 – 0.5 mM ATP, ADP and certain ATP analogues. In this case no increase in DNase I activity occurs, even in the presence of EDTA. At high salt concentrations and in the presence of Mg2+ (‘physiological conditions’) the association rate constants for ATP, ADP and ɛATP (1,N6-ethenoadenosine 5′-triphosphate) and the dissociation rate constant for ɛATP were determined. Both the on and off rates were found to be reduced by a factor about 10 when compared to uncomplexed actin. Thus the binding constant of ɛATP to actin is almost unaltered after complexing to DNase I (2.16 × 108 M−1). Titrating the increase in DNase I activity of the actin – DNase I complex against nucleotide concentration in the presence of EDTA, the association constant of ATP to the cation-free form of actin – DNase I complex was found to be 5 × 103 M−1, which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca2+ to the high-affinity metal-binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca2+ release decreases by a factor of 8 after actin binding to DNase I. The rate of denaturation of nucleotide-free and metal-ion-free actin – DNase I complex was found to be reduced by a factor of about 15. The ATPase activity of the complex is stimulated by addition of Mg2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.