Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling
Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling
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DOI：
10.1074/jbc.m308318200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
Geeves, MA
中科院分区：
文献类型：
--
作者：
Miller, BM;Nyitrai, M;Geeves, MA
通过测量天然存在的以及嵌合的果蝇肌肉肌球蛋白异构体的瞬态动力学参数，研究了肌球蛋白功能的分子机制。我们评估了天然的胚胎异构体、天然的间接飞行肌异构体，以及两种在间接飞行肌和胚胎异构体之间交换了转换结构域的嵌合异构体。从转基因果蝇的间接飞行肌中纯化肌球蛋白，并通过α - 糜蛋白酶消化产生S1。先前对脊椎动物和扇贝肌球蛋白的研究表明，在运动性测定中肌动蛋白丝的速度与横桥解离速率，特别是ADP释放速率之间存在相关性。相比之下，我们的研究表明，在果蝇肌球蛋白异构体中，解离速率与肌动蛋白丝速度之间没有相关性，而且转换结构域对控制肌球蛋白从肌动蛋白上解离的生化动力学没有显著影响。我们认为，对单个肌肉肌球蛋白基因的进化压力可能使所有异构体都保持较快的解离速率。因此，结合速率以及动力冲程的完成，或者肌动蛋白 - 肌球蛋白 - ADP状态之间的平衡，可能决定了这些肌球蛋白异构体的肌动蛋白丝速度。
The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin . myosin . ADP states may define actin filament velocity for these myosin isoforms.