Development of techniques to measure efficiency of energy conversion in the elementary process of actin-myosin sliding

现将1993年至1995年的研究成果总结如下：测定肌动蛋白-肌球蛋白每ATP分子滑动的单位距离：我们用激光闪光光解笼中ATP激活单根甘油肌纤维，并测量光释放ATP的量与肌丝滑动距离的关系。研究发现，当ATP分子数与纤维内肌球蛋白分子数相同时，纤维均匀缩短最小值（约10 nm/半个肌节）。我们还检测了肌凝蛋白涂层玻璃微针与肌动蛋白丝阵列之间由于ATP离子电泳引起的滑动距离，发现肌动蛋白与肌凝蛋白之间的最小滑动距离也在10 nm左右。测量肌肉纤维的化学-机械能转换效率：我们通过激光闪光对笼状Ca进行光解，激活含有与肌凝蛋白头数相等的ATP分子的单甘油肌肉纤维。通过将纤维释放到零力水平，并测量随后的等长力的发展，估计ATP的使用量。结果表明，随着距离的缩短，ATP的使用量成比例地增加，而纤维的功呈钟形，在等长和自由载荷条件下为零。这表明单个肌凝蛋白头部的化学-机械能转换效率在负荷依赖性方面存在很大差异。植物细胞中肌动蛋白-肌球蛋白滑动引起细胞质流动的力-速度关系：我们确定了藻类细胞中肌动蛋白-肌动蛋白滑动的力-速度关系，发现非常高的自由载滑动速度与能量转换效率低有关。

The results obtained by our research work from 1993 to 1995 are summarized as follows.1.Determination of the unitary distance of actin-myosin sliding per ATP molecule : We activated single glycerinated muscle fibers by laser flash photolysis of caged ATP,and measured the relation between the amount of photoreleased ATP and the distance of myofilament sliding. It was found that the minimum uniform fiber shortening (about 10 nm/half sarcomere) was produced when the number of ATP molecules was the same as the number of myosin molecules within the fiber. We also examined the distance of sliding between myosin-coated glass microneedles and actin filament arrays caused by iontophoretic application of ATP,and found that the minimum actin-myosin sliding distance was also about 10 nm.2.Measurement of the efficiency of chemo-mechanical energy conversion in muscle fibers : We activated single glycerinated muscle fibers, which contained ATP molecules equal to the myosin heads in number, by laser flash photlysis of caged Ca. The amount of ATP utilized was estimated by releasing the fiber to zero force level, and measuring the subsequent development of isometric force. The results showed that the amount of ATP utilized increased in proportion to the distance shortened, while the amount of work done by the fiber was bell-shaped, being zero in the isometric and free-loaded conditions. This indicates that the efficiency of chemo-mechanical energy conversion in the individual myosin heads differs greatly in a load-dependent manner.3.Force-velocity relation of actin-myosin sliding causing cytoplasmic streaming in plant cells : We determined the force-velocity relation of actin-myosin sliding in algal cells, and found that the very high free-loaded sliding velocity is associated with low efficiency of energy conversion.