Different Polymeric Forms of Actin Detected by the Fluorescent Probe Terbium Ion

The interaction of actin with Tb3+ was studied by following the fluorescence emitted at 545 nm when the protein was excited at 285 nm in the presence of Tb3+. It was shown that, at low ionic strength, each actin monomer binds, at saturation, six Tb3+ with an association constant of 0.8µM-1. In the presence of 0.1 M KC1 the association constant decreases to 0.15 and 0.24µM-1 at subcritical and overcritical actin concentrations, respectively; the number of the binding sites remains six. When polymeric actin is formed by the addition of 2 mM MgCl2, the association constant drops to 0.008µM-1 and the number of the binding sites to four. The lower number of the Tb3+ binding sites (four) in the actin polymerized by MgCl2 as compared to the number of binding sites (six) of the actin polymerized by KC1 is taken as evidence of a looser structure of this latter polymer. We have also shown that Tb3+ does not replace 45Ca2+ at the single, “high-affinity” site of G-actin. Removal of this Ca2+, in the presence of Tb3+, destroys the typical G- and F-actin structures. © 1981, American Chemical Society. All rights reserved.