Interaction of Actin with Divalent Cations: 2. Characterization of Protein‐Metal Complexes

The interaction of actin with various cations has been studied at pH 7.6, at 20 °C, by means of gel filtration and centrifugation techniques. In addition to the single high‐affinity site, five low‐affinity sites with apparent association constants of 5–6 × 10³ M⁻¹ for Ca²⁺Mn²⁺ and Sr²⁺ and of 1.6 × 10⁴ M⁻¹ for Ni²⁺ have been found in the actin molecule. Unlike the other cations, Zn²⁺ appears to have three high‐affinity sites from which it is not removed by Dowex 50 treatment, and eight low‐affinity sites with an apparent association constant of 1.3 × 10⁴ M⁻¹. Competition experiments suggest that Ca²⁺, Sr²⁺, Mn²⁺ and Ni²⁺ are bound to the same low‐affinity sites, some of which are also available for Zn²⁺. Zn²⁺ also binds to additional sites which are not available for the other cations. Binding of 4 mol of either Ca²⁺, Mn²⁺, Sr²⁺, Ni²⁺ or Zn²⁺ per mol of actin monomer at the low‐affinity sites characterized above causes a complete transformation of the monomeric actin into polymers. The nonidentity of some of the Zn²⁺‐binding sites with those for the other cations seems to explain structural differences between the polymers formed in the presence of this cation and those produced upon binding of Mn²⁺, Ni²⁺ or alkaline earth metals, described in the preceding paper in this journal. The results suggest that formation of the double‐stranded F‐actin filaments requires the neutralization of negative charge at specific sites in the monomer molecules. The aggregation of F‐actin filaments into paracrystals observed at pH 7.6 in the presence of Ca²⁺, Mg²⁺, Sr²⁺ or Mn²⁺ at millimolar concentrations seems to involve the binding of these cations to a third class of binding sites with apparent association constants of the order of 10² M⁻¹. The binding parameters to this class of sites could not be determined by the methods used in this work.