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 x 10³ M^-1 for Ca²⁺, Mn²⁺ and Sr²⁺ and of 1.6 x 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 x 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 ²⁺-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. [ABSTRACT FROM AUTHOR]