The binding of myosin subfragment-1 to F-actin in the absence of nucleotide. Evidence for dependence on F-actin concentration.

The binding of myosin subfragment-1 (S-1) to F-actin in the absence of nucleotide was examined by the sedimentation method using 1,5-IAEDANS-labeled S-1. We found that the binding affinity of F-actin to S-1 was dependent on the concentration of F-actin, and the binding was weaker at higher concentrations of F-actin. The apparent association constant determined from a linearly extrapolated Scatchard plot was 6.5 x 10(6) M-1 at 8.1 microns F-actin, and 1.7 x 10(7) M-1 at 2.0 microns F-actin in 120 mM KC1, 2 mM MgCl2, 0.1 mM CaCl2, and 20 mM Tris-acetate (pH 7.6) at 20 degrees C. Furthermore, the Scatchard plot revealed the existence of cooperativity in the binding of S-1 to F-actin. In order to obtain higher precision we developed a new method for the chromatographic determination of free S-1 in acto-S-1 solution. By this method we could determine free S-1 concentrations of the order of 10(-9) M easily and accurately. The above conclusion obtained by the sedimentation method was confirmed by this chromatographic method, and these effects can be well explained by considering the length distribution of F-actin. We propose an allosteric model in which both the length distribution and the polarity of F-actin are taken into consideration.