The dissociation constant of the actin-heavy meromyosin subfragment-1 complex.

We measured the binding of [14C]iodoacetamide labeled heavy meromyosin subfragment-1 (S-1) to F-actin by sedimenting the actin-S-1 complex and assaying the radioactivity remaining in the supernatant. The apparent dissociation constants (Kd) at 25 degrees, pH 7.0, were 0.01 to 0.04 muM at 0.027 and 0.08 ionic strengths and 0.07 to 0.14 muM at 0.14 ionic strength. Kd was not altered when the troponin-tropomyosin complex was bound on the actin, nor was it affected by free calcium concentration in the range 10(-4) to 10(-9) M. Measurements of the displacement of labeled S-1 from actin by native S-1 showed labeling had not altered Kd. In control experiments we found that at the low actin concentrations used (0.001-0.5 muM) not all of the actin sedimented and, furthermore, the data suggested that some of the S-1 in the supernatant was bound to supernatant actin. Our estimation of Kd, based on the assumption that all the supernatant S-1 was free, therefore resulted in an apparent Kd greater than the true Kd. We minimized the effect of the supernatant actin artefact by using only the data for high ratios of S-1 to actin, where no less than 75% of the actin sedimented; we estimate that the true Kd values could not be less than half the apparent Kd values.