Single molecule analysis on the FENN effect of actomyosin motor

肌动球蛋白运动的FENN效应的单分子分析

• 批准号: 10680641
• 负责人: CHAEN Shigeru
• 金额: $ 2.11万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680641/
• 关键词: Actomyosin; FENN effect; single molecule process; motor protein

In order to study the FENN effect of actomyosin motor, we have been studying the nucleotide exchange rate constants between the bound nucleotide and ATP on flash photolysis of caged ATP during shortening of rabbit psoas myofibrils, and shown the strain-dependent rate constant during the shortening(chaen et al. 1997, Biophys. J). To ascertain the above result, we have measured the rate constant of soleus myofibrils, which is expected to be better preparation to our method limited by video rate (30 Hz). When the myofibril was allowed to shorten, the rate constant was increased with increasing shortening velocity, while the rate constant was not significantly affected by stretch. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. Moreover, to extend this study from myofibril to actomyosin motor, we have constructed an apparatus to examine if the lifetime of nucleotide bound state depends on the strain of the cross-bridge. The apparatus consists of total internal reflection fluorescence microscopy, dual view imaging unit, and image-intensified CCD camera. By tracing the image of the fluorescent nucleotide on a video frame with scanning mirror, we have succeeded in measuring the shorter life time than 1/30 sec in the ordinary video system (30 frames /sec).

为了研究肌动球蛋白马达的FENN效应，我们已经研究了在兔腰肌肌原纤维缩短期间结合的核苷酸和ATP对笼状ATP的闪光光解的核苷酸交换速率常数，并且显示了缩短期间的应变依赖性速率常数（chaen等人，1997，Biophys. J）。为了确定上述结果，我们测量了比目鱼肌肌原纤维的速率常数，这有望为我们的视频速率限制（30 Hz）的方法做好准备。当肌原纤维被允许缩短时，速率常数随着缩短速度的增加而增加，而速率常数不受拉伸的显著影响。这些结果表明，跨桥动力学没有显着影响，在较高的应变在延长，但取决于较低的应变在缩短。此外，为了将这项研究从肌原纤维扩展到肌动球蛋白马达，我们构建了一个装置来检查核苷酸结合态的寿命是否取决于横桥的应变。该装置由全内反射荧光显微镜、双视角成像单元和图像增强CCD摄像机组成。通过用扫描镜在视频帧上跟踪荧光核苷酸的图像，我们成功地测量了在普通视频系统（30帧/秒）中小于1/30秒的寿命。

项目成果

Ibuki Shirakawa, Shigeru Chaen, Clive R. Bagshaw and Haruo Sugi: "Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog"Biolphysical Journal. 78. 918-926 (2000

Ibuki Shirakawa、Shigeru Chaen、Clive R. Bagshaw 和 Haruo Sugi：“使用荧光 ATP 类似物测量单个兔子比目鱼肌原纤维在缩短和延长过程中的核苷酸交换率常数”生物物理学杂志。

Ibuki Shirakawa: "Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog."Biophysical Journal. 78. 918-926 (2000)

Ibuki Shirakawa：“使用荧光 ATP 类似物测量单个兔子比目鱼肌原纤维在缩短和延长过程中的核苷酸交换率常数。”生物物理学杂志。

Shigeru Chaen: "Measurement of ATP turnover during shortening and lengthering of rabbit psoas myofibrils using a fluorescent ATP analog." Adv.Exp.Med.Biol.453. 569-576 (1998)

Shigeru Chaen：“使用荧光 ATP 类似物测量兔腰肌肌原纤维缩短和延长过程中的 ATP 周转率。”

Ibuki Shirakawa: "Measurement of mucleotide exchange note constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog."Biophysical Journal. 78. 918-926 (2000)

Ibuki Shirakawa：“使用荧光 ATP 类似物测量单个兔子比目鱼肌原纤维在缩短和延长过程中的核苷酸交换常数。”生物物理学杂志。

Hidetake Miyata: "Evanescent excitation microscopy .Its application to the study of single molecular process kinetics of actomyosin motor." Adv.Exp.Med.Biol.453. 29-35 (1998)

Hidetake Miyata：“倏逝激发显微镜。其在肌动球蛋白运动的单分子过程动力学研究中的应用。”