Carlier M F, Pantaloni D
Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
J Biol Chem. 1988 Jan 15;263(2):817-25.
Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.
我们之前的工作（卡利尔，M.-F.，和潘塔洛尼，D.（1986年）《生物化学》25卷，7789 - 7792页）表明，F-ADP-Pi-肌动蛋白是ATP-肌动蛋白聚合过程中的主要中间体，这是由于在细丝上ATP裂解后Pi释放的速率较慢。为了理解ATP-肌动蛋白聚合的机制，我们制备了F-ADP-Pi-肌动蛋白并对其动力学参数进行了表征。32Pi以1摩尔/摩尔F-肌动蛋白亚基的化学计量比与F-ADP-肌动蛋白结合，在pH 7.0时平衡解离常数Kpi为1.5 mM。Kpi随pH升高而增加，表明H2PO-4物种与F-肌动蛋白结合。ADP-Pi-肌动蛋白亚基从细丝末端解离的速度比ADP-肌动蛋白亚基慢得多；因此，细丝在ATP中的稳定性归因于末端的ADP-Pi亚基。ADP-Pi-肌动蛋白解离速度慢也解释了里卡德和谢特林（理查德，J. E.，和谢特林，P.（1986年）《分子生物学杂志》191卷，273 - 280页）报道的在有Pi存在时ADP-肌动蛋白临界浓度的降低。Pi对肌动蛋白从细丝上解离速率的影响在带刺末端比在 pointed 末端更明显。使用凝溶胶蛋白封闭带刺末端，我们已经表明在存在ATP和饱和Pi的情况下，两端在能量上是不同的，但比在没有Pi的情况下差异小。这些结果在一个新的肌动蛋白聚合模型中得到了解释。磷酸盐与F-肌动蛋白的结合可能调节肌肉和非肌肉细胞中的运动事件。
