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Dissociation of Acto-H-Meromyosin and That of Acto-Subfragment-1 Induced by Adenyl-5′-yl-Imidodiphosphate: Evidence for a Ternary Complex of F-Actin, Myosin Head, and Substrate1

1980; Oxford University Press; Volume: 88; Issue: 6 Linguagem: Inglês

10.1093/oxfordjournals.jbchem.a133140

1756-2651

Akio Inoue, Yūji Tonomura,

Force Microscopy Techniques and Applications

Acto-HMM or acto-S-1 was dissociated by the addition of AMIPPNP in 50 nmt KCl, 2 mM MgCl2 and 10 mM Tris-HCl at pH 7.8 and 20°C, and the extent of dissociation of HMM or S-1 from acto-HMM or acto-S-1, α, was measured by a light-scattering method. The dependence of α on the concentrations of F-actin and of AMPPNP was found to be given by the equation α=l/[1 +[A] (1 +Ka/[S]/Ka], where [A] and [S] are the concentrations of actin monomer and AMPPNP, respectively. This equation suggested the existence of a ternary complex of F-actin, HMM or S-1, and AMPPNP, since α approached 0 when the F-actin concentration was increased even in the presence of high concentrations of AMPPNP. The dissociation constant of a complex of HMM-AMPPNP with F-actin, Ka., and that of a complex of acto-HMM with AMPPNP, KKa, were estimated to be 5.7±1.5 and 850±200 μM, respectively, for four different preparations. When S-1 was used in place of HMM, the Ka value increased 2–3 times, while the Ka value remained unchanged. The rate constant for dissociation of acto-HMM induced by AMPPNP was determined under conditions where α was nearly 1. The time course of decrease in the light-scattering intensity, ΔLS, after adding AMPPNP to acto-HMM was found to be given by the relationship ΔLS=ΔLSmax (1-e−vrt), where ΔLSmax is ΔLS at t=∞ i.e., atα⋍1.The dependence on AMPPNP concentration, [S] of the apparent first-order rate constant, Vt was found to be given by vt=Ka/(1+ka/[S]). The values of ka and ka were 2 s−1 and 810 μM, respectively. The rate constant for binding of F-actin with an HMM-AMPPNP complex was determined under conditions where α was nearly 0. The time course of increase in the light-scattering intensity, ALS, after adding a sufilciently high concentration of F-actin to HMM-AMPPNP complex was found to be given by the relationship ΔLS=ΔLSmax (1-e−vrt, where ΔLSmax is Δat t=∞, i.e., at α=0. The apparent first-order rate constant, vr was found to be given by Vr=k-a [A] and the k-a value was found to be 0.12 μM−1∣S−1. The ratio, ka/k-a was almost equal to the Ka value obtained by measuring the dependence of α on [A] and [S]. The dependence of α of acto-HMM on the concentrations of F-actin and AMPPNP was also determined in the presence of 2 mi MnCl2 by the light-scattering method. The values of Ka and K8 were found to be 5.8 and 60 μM respectively. The binding of AMPPNP to HMM in the presence of F-actin was directly measured by an ultracentrifugal separation method at concentrations of F-actin much higher than Ka We found that the extent of AMPPNP binding was independent of the F-actin concentration, and that AMPPNP bound to each head of HMM with a dissociation constant of 60 μM.