Brenner S L, Korn E D
J Biol Chem. 1983 Apr 25;258(8):5013-20.
The rate of exchange of G-actin with subunits of F-actin and the rate of hydrolysis of ATP in solutions of F-actin at steady state have been measured simultaneously. Subunit exchange kinetics were analyzed by both a treadmill model and an exchange-diffusion model. The best fit to a treadmill model of the data obtained in 0.5 mM MgCl2 and 0.2 mM ATP at 30 degrees C gave a treadmill efficiency (net monomers incorporated per ATP hydrolyzed) of 0.26, in good agreement with the previously reported s-value of 0.25 (Wegner, A. (1976) J. Mol. Biol. 108, 139-150) for similar ionic conditions. However, in this and other conditions with excess free divalent cations (Ca2+ or Mg2+), the observed exchange kinetics were in better agreement with an exchange-diffusion model than with a treadmilling model over the entire time course of the experiment. In the absence of excess divalent cations (50 mM KCl), exchange was too slow to be analyzed adequately by either model. Using the measured filament length distribution and the observed fit of the exchange-diffusion model to the data in 0.5 mM MgCl2, an on-rate constant of 2.8 x 10(6) M-1 S-1 and an off-rate constant of 5.8 s-1 were calculated. These values, while in good agreement with previously measured pre-steady state polymerization rate constants under different ionic conditions (Pollard, T. D., and Mooseker, M. S. (1981) J. Cell Biol. 88, 654-659), are about 30-fold higher than the rate constants predicted from the rate of ATP hydrolysis at steady state. To rationalize these discrepancies, a model is proposed in which a segment of F-actin subunits at one or both ends of the filament contains bound ATP at steady state.
已同时测量了G-肌动蛋白与F-肌动蛋白亚基的交换速率以及稳态下F-肌动蛋白溶液中ATP的水解速率。通过跑步机模型和交换扩散模型对亚基交换动力学进行了分析。在30℃下，对0.5 mM MgCl2和0.2 mM ATP条件下获得的数据进行跑步机模型拟合，得到的跑步机效率（每水解一个ATP掺入的净单体数）为0.26，与先前报道的在类似离子条件下的s值0.25（Wegner, A. (1976) J. Mol. Biol. 108, 139 - 150）非常吻合。然而，在这种以及其他存在过量游离二价阳离子（Ca2+或Mg2+）的条件下，在整个实验时间过程中，观察到的交换动力学与交换扩散模型的吻合度优于跑步机模型。在没有过量二价阳离子（50 mM KCl）的情况下，交换太慢，无法用任何一个模型进行充分分析。利用测量的丝状体长度分布以及观察到的交换扩散模型对0.5 mM MgCl2数据的拟合，计算出结合速率常数为2.8×10(6) M-1 S-1，解离速率常数为5.8 s-1。这些值虽然与先前在不同离子条件下测量的预稳态聚合速率常数（Pollard, T. D., and Mooseker, M. S. (1981) J. Cell Biol. 88, 654 - 659）非常吻合，但比根据稳态下ATP水解速率预测的速率常数高约30倍。为了解释这些差异，提出了一个模型，其中丝状体一端或两端的一段F-肌动蛋白亚基在稳态下含有结合的ATP。
