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Abstract:
Equilibrium constants for the formation of a ternary complex between actin, myosin subfragment 1 (S1) and the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate (Ado PP[NH]P) were determined from light-scattering titrations under a variety of conditions. The affinities of S1 (binding constant K1) and acto . S1 (K4) for AdoPP[NH]P have relatively low dependencies on temperature (delta H degrees approximately equal to - 15 - 30 kJ mol-1) and ionic strength, in contrast to the affinities of S1 (K2) and S1 . AdoPP[NH]P (K3) for actin which are influenced quite strongly by temperature (delta H degrees approximately equal to 50 - 65 kJ mol-1) and ionic strength, K2 decreasing by a factor of 10 - 15 between I = 0.05 M and I = 0.2 M and K3 decreasing by a factor of 5.K1, and by detailed balance K2 as well, were found to be about 10-times higher than hitherto reported values (K1 = 3.4 X 10(7) M-1, K2 = 6 X 10(8) M-1, at 24 degrees C,I = 0.09 M, pH 8.0). The binding of ADP to S1 is about 10-fold weaker than that of AdoPP[NH]P, being however much more exothermic (delta H degrees = - 70 kJ mol-1 at I = 0.1 M) and having a negative standard entropy change (delta S = - 125 J mol-1 K-1), in contrast to AdoPP[NH]P binding for which the calculated delta S had positive values. The observed rate constant of dissociation of acto . S1 by AdoPP[NH]P showed an almost hyperbolic dependence on the nucleotide concentration, reaching a maximum of 15 s-1 at I = 0.055 M and 5 s-1 at I = 0.275 M, pH 8.0, 23 degrees C; at 5 degrees C this value was somewhat higher. The rate constant of dissociation of AdoPP[NH]P from its complex with acto . S1 was estimated to exceed 400 s-1 at 23 degrees C, and to be of the order of 150 s-1 at 4 degrees C. The observed rate constant for the association of the S1 . nucleotide complex and actin was proportional to actin concentrations up to 60 microM, thus defining an apparent second-order rate constant of 2 X 10(4) M-1 s-1 at I = 0.125 M and 23 degrees C. A reaction scheme is proposed in which isomerizations of the acto . S1 and acto . S1 . nucleotide complexes can occur.
