Abstract
The bindings of S-1 and the two heads of HMM with pyrene-labeled F-actin were studied using the change in light-scattering intensity or that in the fluorescence intensity of the pyrenyl group. At low ionic strength (50 mM KC1), both S-1 and HMM became bound tightly with F-actin (Kd, ⟨0.1μM) and both heads of HMM became bound to F-actin. The affinities of S-1 and HMM for F-actin decreased with increasing KC1 concentration. In 1 M KC1, the Kd, values of S-1 and HMM for F-actin were 11 and 0.58μM, respectively. Thus, HMM was bound to F-actin 19 times more tightly than S-1. We compared the extent of binding of HMM to F-actin measured by a centrifugation method with that measured by the fluorescence change of pyrenyl-group, and found that even in 1 M KC1, HMM became bound to F-actin with a two-headed attachment. We measured the kinetics of binding and dissociation of acto-S-1 and acto-HMM from the time course of the change in light-scattering intensity after mixing S-1 or HMM with F-actin at 1 M KC1 and that after mixing 1 M KC1 with acto-S-1 or acto-HMM formed at low ionic strength. The results could be explained by the following schemes:S-1+F-actin k1_??_k-1S-1-F-atin
HMM+F-actin k1_??_k-1HMM-F-actin (single-headed) k2k-2HMM=F-actin (double-headed)
where the values of k1 and k-1 for S-1 were 0.13 s-1/μM and 1.8 s-1, respectively. The values of k1 and k-1, for HMM were 0.45 s-1/μM and 1.8 s-1, respectively. The value of k-2/k2 was 0.15 and k-2⟩3s-1 (if k-2=3.6 s-1, k2=24 s-1). Thus, the step from HMM-F-actin (single-headed binding) to HMM=F-actin (double-headed binding) does not depend on F-actin concentration, and the equilibrium of this step favors the double-headed binding.
