Mechanism for nucleotide exchange in monomeric actin

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Abstract
Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (.epsilon.-ADP), or 1,N6-ethenoadenosine triphosphate (.epsilon.-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by .epsilon.-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20.degree. C of the fluorescence difference between bound and free .epsilon.-ATP or .epsilon.-ADP. In the presence of low (< 10 .mu.M) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (> 50 .mu.M), low ADP concentrations displace .epsilon.-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of .epsilon.-ATP by ATP in the presence of either Ca2+or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP. It is concluded that, in general, the presence of Ca2+ strengthens ATP binding relative to ADP, while the presence of Mg2+ weakens ATP binding relative to ADP.