Greene L E, Eisenberg E
J Biol Chem. 1980 Jan 25;255(2):543-8.
The ability of adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP, and PPi to dissociate the actin.myosin subfragment 1 (S-1) complex was studied using an analytical ultracentrifuge with UV optics, which enabled the direct determination of the dissociated S-1. At mu = 0.22 M, pH 7.0, 22 degrees C, with saturating nucleotide present, ADP weakens the binding of S-1 to actin about 40-fold (K congruent to 10(5) M-1), while both AMP-PNP and PPi weakens the binding about 400-fold (K congruent to 10(4) M-1). This 10-fold stronger dissociating effect of AMP-PNP and PPi compared to ADP correlates with our data showing that the binding of AMP-PNP and PPi to S-1 is about 10-fold stronger than the binding of ADP. In contrast, the binding constants of ADP, AMP-PNP, and PPi to acto.S-1 are nearly identical (K congruent to 5 x 10(3) M-1). At 4 degrees C, AMP-PNP has only a 3-fold stronger dissociating effect than ADP and, similarly, our data suggest that the binding of AMP-PNP and ADP to S-1 is quite similar at 4 degrees C. AMP-PNP and PPi are, therefore, somewhat better dissociating agents than ADP, but the difference among these three ligands is quite small. These data also show that actin and nucleotide bind to separate but interacting sites on S-1 and that the S-1 molecules bind independently along the F-actin filament with a binding constant of about 1 x 10(7) M-1 at 22 degrees C and physiological ionic strength.
利用配备紫外光学系统的分析超速离心机研究了腺苷 - 5'-亚氨基二磷酸（AMP - PNP）、ADP和焦磷酸（PPi）解离肌动蛋白 - 肌球蛋白亚片段1（S - 1）复合物的能力，该系统能够直接测定解离后的S - 1。在μ = 0.22 M、pH 7.0、22℃且存在饱和核苷酸的条件下，ADP使S - 1与肌动蛋白的结合减弱约40倍（K约为10⁵ M⁻¹），而AMP - PNP和PPi均使结合减弱约400倍（K约为10⁴ M⁻¹）。与ADP相比，AMP - PNP和PPi的解离作用强10倍，这与我们的数据相符，即AMP - PNP和PPi与S - 1的结合比ADP与S - 1的结合强约10倍。相反，ADP、AMP - PNP和PPi与肌动蛋白 - S - 1的结合常数几乎相同（K约为5×10³ M⁻¹）。在4℃时，AMP - PNP的解离作用仅比ADP强3倍，同样，我们的数据表明在4℃时AMP - PNP和ADP与S - 1的结合相当相似。因此，AMP - PNP和PPi作为解离剂比ADP稍好，但这三种配体之间的差异相当小。这些数据还表明，肌动蛋白和核苷酸结合到S - 1上不同但相互作用的位点，并且在22℃和生理离子强度下，S - 1分子沿着F - 肌动蛋白丝独立结合，结合常数约为1×10⁷ M⁻¹。
