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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 x 10(8) 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 x 10(3) 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.
