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

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Abstract
The stoichiometric actin-DNase-I complex was used to study the actin-nucleotide and actin-divalent-cation 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 .epsilon.ATP (1,N6-ethenoadenosine 5''-triphosphate) and the dissociation rate constant for .epsilon.ATP were determined. Both the on and off rates were found to be reduced by a factor of about 10 when compared to uncomplexed actin. Thus the binding constant of .epsilon.ATP to actin is almost unaltered after complexing to DNase I (2.16 .times. 108 M-1). Titrating the increase in DNaseI 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 .times. 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.