Konrad M, Goody R S
Eur J Biochem. 1982 Nov 15;128(2-3):547-55. doi: 10.1111/j.1432-1033.1982.tb07000.x.
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.
在多种条件下，通过光散射滴定法测定了肌动蛋白、肌球蛋白亚片段1（S1）与不可水解的ATP类似物腺苷5'-[β,γ-亚氨基]三磷酸（AdoPP[NH]P）形成三元复合物的平衡常数。与S1（结合常数K1）和肌动蛋白·S1（K4）对AdoPP[NH]P的亲和力相比，它们对温度（ΔH°约等于-15 - 30 kJ·mol⁻¹）和离子强度的依赖性相对较低。相反，S1（K2）和S1·AdoPP[NH]P（K3）对肌动蛋白的亲和力受温度（ΔH°约等于50 - 65 kJ·mol⁻¹）和离子强度的影响很大，K2在I = 0.05 M和I = 0.2 M之间降低10 - 15倍，K3降低5倍。发现K1以及通过详细平衡得到的K2约比迄今报道的值高10倍（在24°C、I = 0.09 M、pH 8.0时，K1 = 3.4×10⁷ M⁻¹，K2 = 6×10⁸ M⁻¹）。ADP与S1的结合比AdoPP[NH]P弱约10倍，然而放热得多（在I = 0.1 M时，ΔH° = -70 kJ·mol⁻¹）且标准熵变为负值（ΔS = -125 J·mol⁻¹·K⁻¹），这与AdoPP[NH]P结合的计算结果相反，其ΔS为正值。观察到的AdoPP[NH]P使肌动蛋白·S1解离的速率常数对核苷酸浓度呈现几乎双曲线的依赖性，在I = 0.055 M、pH 8.0、23°C时达到最大值15 s⁻¹，在I = 0.275 M时为5 s⁻¹；在5°C时该值略高。在23°C时，AdoPP[NH]P从其与肌动蛋白·S1的复合物中解离的速率常数估计超过400 s⁻¹，在4°C时约为150 s⁻¹。观察到的S1·核苷酸复合物与肌动蛋白结合的速率常数与肌动蛋白浓度成正比，直至60 μM，因此在I = 0.125 M和23°C时定义了一个表观二级速率常数为2×10⁴ M⁻¹·s⁻¹。提出了一个反应方案，其中肌动蛋白·S1和肌动蛋白·S1·核苷酸复合物可能发生异构化。
