HIGH-AFFINITY BINDING OF DIVALENT-CATION TO ACTIN MONOMER IS MUCH STRONGER THAN PREVIOUSLY REPORTED

Monomeric actin is known to bind tightly one divalent cation per molecule. We have quantitatively reinvestigated the affinity of actin for Ca2+ and Mg2+ using the fluorescent Ca2+ chelator Quin2 to induce and measure the dissociation of Ca2+ from Ca-actin, supporting these studies with measurements using 45Ca. We found that the KD for Ca-actin is actually 1.9 .+-. 0.7 nM. Kinetic analysis supported this result and demonstrated a dissociation rate constant (k-) of 0.013 s-1 and an association rate constant (k+) of 6.8 x 106 M-1 s-1 for Ca-actin. Competitive binding studies indicated that the binding affinity of actin for Ca2+ is 5.4 times that for Mg2+, yielding a calculated KD for Mg-actin of about 10 nM. Thus, the tight-binding of divalent cations to actin is 3-4 orders of magnitude stronger than previously thought.