An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle
An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle
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DOI：
10.1113/jphysiol.2011.222984
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发表时间：
2012-02-01
影响因子：
5.5
通讯作者：
Piazzesi, G.
中科院分区：
文献类型：
--
作者：
Elangovan, R.;Capitanio, M.;Piazzesi, G.
一种从青蛙骨骼肌中提取和保存肌球蛋白II的新的有效方法使肌球蛋白的功能保存一周成为可能，并将单分子技术应用于分子马达，该分子马达的机械、结构和能量参数得到了最好的原位表征。通过体外运动试验，我们估计了肌动蛋白在蛙肌球蛋白II(VF)上的滑动速度以及pH、肌球蛋白密度、温度(4-30℃)和底物浓度对其的调节作用。Vf在30.6℃时为8.88±0.26µms-1，在4.5℃时降至1.60±0.09µms-1。体外力学和动力学参数与蛙肌肌球蛋白在每半肌节上排列的原位参数相结合。通过将Vf与不同载荷下测得的完整蛙肌纤维的缩短速度及其与温度的关系进行比较，我们发现在相同温度下，Vf比未加载纤维的缩短速度(V0)小40-50%，并确定了解释Vf减小值的载荷。利用这一综合方法，我们可以在原位确定acto-myosin ATPase循环的基本动力学步骤及其与机械步骤的关系。特别是，我们发现，在5摄氏度时，使用从现场实验估计的步长计算的ADP释放速度解释了在低负荷下稳定缩短过程中发动机的脱离速度。
A new efficient protocol for extraction and conservation ofmyosin II fromfrog skeletal muscle made it possible to preserve themyosin functionality for a week and apply singlemolecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4-30 degrees C) and substrate concentration. VF was 8.88 +/- 0.26 mu ms-1 at 30.6 degrees C and decreased to 1.60 +/- 0.09 mu ms-1 at 4.5 degrees C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40-50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5 degrees C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads.