Open AccessThe complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin
Author(s) -
POLZAR Bernhard,
NOWAK Ewa,
GOODY Roger S.,
MANNHERZ Hans Georg
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14826.x
Subject(s) - nucleotide , cytochalasin , deoxyribonuclease i , chemistry , divalent , actin , biophysics , actina , biochemistry , dissociation constant , cytoskeleton , biology , receptor , dna , organic chemistry , gene , base sequence , cell
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 MgCl 2 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 Mg 2+ (‘physiological conditions’) the association rate constants for ATP, ADP and ɛATP (1, N 6 ‐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 × 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 × 10 3 M −1 , which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca 2+ to the high‐affinity metal‐binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca 2+ 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 Mg 2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.