Actin and light chain isoform dependence of myosin V kinetics
Actin and light chain isoform dependence of myosin V kinetics
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DOI：
10.1021/bi001701b
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发表时间：
2000-11-21
期刊：
影响因子：
2.9
通讯作者：
Ostap, EM
中科院分区：
文献类型：
--
作者：
De la Cruz, EM;Wells, AL;Ostap, EM
近期关于肌球蛋白V的研究报道了一些动力学差异，这些差异可能归因于所使用的不同重链（鸡与小鼠）和轻链（必需轻链与钙调蛋白）异构体。了解单个轻链异构体对肌球蛋白V动力学行为的影响程度至关重要，因为尚不清楚细胞中哪些轻链与肌球蛋白V结合。此外，到目前为止的所有研究都使用了α -骨骼肌肌动蛋白，而肌球蛋白V存在于表达β -和γ -肌动蛋白的非肌肉细胞中。因此，我们对单头肌球蛋白V动力学的肌动蛋白和轻链依赖性进行了表征。当结合两种必需轻链异构体中的任何一种或钙调蛋白时，由运动结构域和第一个轻链结合结构域组成的肌球蛋白V构建体的最大肌动蛋白激活的稳态ATP酶速率（V-max）是相同的。然而，当结合钙调蛋白时，K - ATP酶显著更高，并且ATP水解的速率常数和平衡常数降低，这表明必需轻链有利于M·ADP·P - i状态的形成。肌球蛋白V的动力学参数不受肌动蛋白异构体的强烈影响。在所有肌动蛋白和轻链异构体的ATP酶循环中，ADP从肌动蛋白 - 肌球蛋白复合物中的释放是限速步骤。我们推测，尽管在动力学中存在显著的依赖轻链的改变，这些改变可能会影响体外测定中肌球蛋白V的持续性，但在生理条件下这些差异可能会最小化。
Recent studies on myosin V report a number of kinetic differences that may be attributed to the different heavy chain (chicken vs mouse) and light chain (essential light chains vs calmodulin) isoforms used. Understanding the extent to which individual light chain isoforms contribute to the kinetic behavior of myosin V is of critical importance, since it is unclear which light chains are bound to myosin V in cells. In addition, all studies to date have used alpha -skeletal muscle actin, whereas myosin V is in nonmuscle cells expressing beta- and gamma -actin. Therefore, we characterized the actin and light chain dependence of single-headed myosin V kinetics. The maximum actin-activated steady-state ATPase rate (V-max) of a myosin V construct consisting of the motor domain and first light chain binding domain is the same when either of two essential light chain isoforms or calmodulin is bound. However, with bound calmodulin, the K-ATPase is significantly higher and there is a reduction in the rate and equilibrium constants for ATP hydrolysis, indicating that the essential light chain favors formation of the M.ADP.P-i state. No kinetic parameters of myosin V are strongly influenced by the actin isoform. ADP release from the actin-myosin complex is the rate-limiting step in the ATPase cycle with all actin and light chain isoforms. We postulate that although there are significant light-chain-dependent alterations in the kinetics that could affect myosin V processivity in in vitro assays, these differences likely are minimized under physiological conditions.