Divalent Cation Binding to the High- and Low-Affinity Sites on G-Actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using ⁴⁵Ca²⁺ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N’-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca²⁺ affinity: a single high-affinity site with a K_d considerably less than 1 μM and three identical moderate-affinity binding sites with a K_d of 18 μM. The data for the Mg²⁺-induced fluorescence enhancement of actin labeled with N-acetyl-N’-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882–2886] in which Ca²⁺ is replaced by Mg²⁺ at the moderate affinity sites(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca²⁺ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca²⁺ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg²⁺ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd²⁺ or Mn²⁺ instead of Mg²⁺. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses. This method allows testing of different kinetic mechanisms by statistical methods and has been used in the fitting of actin isomerization data.