Comparative kinetic and functional characterization of the motor domains of human nonmuscle myosin-2C isoforms.
Comparative kinetic and functional characterization of the motor domains of human nonmuscle myosin-2C isoforms.
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DOI：
10.1074/jbc.m110.212290
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发表时间：
2011-06-17
期刊：
影响因子：
--
通讯作者：
Manstein DJ
中科院分区：
文献类型：
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作者：
Heissler SM;Manstein DJ
非肌肉肌球蛋白广泛分布，在维持细胞形态和胞质分裂中起重要作用。在本研究中，我们比较了人非肌肉肌球蛋白重链（NMHC）-2C运动结构域天然存在的转录变体的详细动力学和功能特性。NMHC - 2C在环1和环2中均存在可变剪接。异构体2C0在两个环中均无插入片段，是最短的异构体。异构体2C1和2C1C2在环1区域有一个8个氨基酸的延伸。异构体2C1C2在环2区域另外还有一个33个氨基酸的延伸。瞬态动力学实验表明，在有核苷酸和无核苷酸的情况下，异构体2C1C2与F - 肌动蛋白结合的速率常数增加，这可归因于环2的延伸。ADP结合在三种转录变体中仅显示出微小差异。相比之下，在有F - 肌动蛋白和无F - 肌动蛋白的情况下，ADP释放速率均观察到较大差异。在异构体2C0和2C1C2之间观察到的差异最大。在有F - 肌动蛋白和无F - 肌动蛋白的情况下，异构体2C1C2的ADP亲和力提高了5 - 7倍。此外，我们的结果表明，所有三种异构体的ADP释放动力学都受到游离Mg²⁺离子浓度变化的调节。在0.2 - 1.5 mM游离Mg²⁺离子的生理范围内，观察到NMHC - 2C异构体的最大响应性，这影响它们的工作比、速度和承载张力的特性。
Nonmuscle myosins are widely distributed and play important roles in the maintenance of cell morphology and cytokinesis. In this study, we compare the detailed kinetic and functional characterization of naturally occurring transcript variants of the motor domain of human nonmuscle myosin heavy chain (NMHC)-2C. NMHC-2C is alternatively spliced both in loop-1 and loop-2. Isoform 2C0 contains no inserts in either of the loops and represents the shortest isoform. An 8-amino acid extension in the loop-1 region is present in isoforms 2C1 and 2C1C2. Isoform 2C1C2 additionally displays a 33-amino acid extension in the loop-2 region. Transient kinetic experiments indicate increased rate constants for F-actin binding by isoform 2C1C2 in the absence and presence of nucleotide, which can be attributed to the loop-2 extension. ADP binding shows only minor differences for the three transcript variants. In contrast, larger differences are observed for the rates of ADP release both in the absence and presence of F-actin. The largest differences are observed between isoforms 2C0 and 2C1C2. In the absence and presence of F-actin, isoform 2C1C2 displays a 5–7-fold increase in ADP affinity. Moreover, our results indicate that the ADP release kinetics of all three isoforms are modulated by changes in the concentration of free Mg2+ ions. The greatest responsiveness of the NMHC-2C isoforms is observed in the physiological range from 0.2 to 1.5 mm free Mg2+ ions, affecting their duty ratio, velocity, and tension-bearing properties.