Single molecule analysis on the FENN effect of actomyosin motor

为了研究肌动球蛋白马达的Fenn效应，我们研究了在兔腰肌肌原纤维缩短过程中，结合核苷酸与ATP之间的核苷酸交换速率常数对笼养ATP闪光降解的影响，并显示了缩短过程中与应变有关的速率常数(Chaen等人)。1997年，生物物理学。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，以期更好地准备我们的方法限制视频速率(30赫兹)。允许肌原纤维缩短时，速率常数随缩短速度的增加而增大，而拉伸对速率常数的影响不明显。这些结果表明，在拉长过程中，较高应变对过桥动力学没有显著影响，而在拉长过程中，则依赖于较低应变。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状态的寿命是否依赖于跨桥的应变。该装置由全内反射荧光显微镜、双视成像单元和像增强型CCD摄像机组成。通过用扫描镜跟踪视频帧上的荧光核苷酸图像，我们成功地测量了比普通视频系统中的1/30秒更短的寿命(30帧/秒)。

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).