VanBuren P, Guilford W H, Kennedy G, Wu J, Warshaw D M
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington 05405, USA.
Biophys J. 1995 Apr;68(4 Suppl):256S-258S; 258S-259S.
Smooth muscle generates as much force per cross sectional area of muscle as skeletal muscle with only one-fifth the myosin content. Although this apparent difference could be explained at the tissue or cellular level, it is possible that at the molecular level smooth muscle cross-bridges generate greater average force than skeletal muscle cross-bridges. To test this hypothesis, we used an in vitro motility assay (VanBuren et al., 1994) in which either chicken thiophosphorylated gizzard smooth or pectoralis skeletal muscle monomeric myosin is adhered to a nitrocellulose surface. A fluorescently labeled actin filament, attached to an ultracompliant (50-200 nm/pN) glass microneedle, is brought in contact with the myosin surface. Isometric force, being generated by myosin cross-bridges pulling on the attached actin filament, is calculated from the extent to which the calibrated microneedle is deflected. By measuring the density of myosin adhered to the surface, we estimated the number of myosin cross-bridges that are able to interact with a length of actin filament in contact with the myosin surface. In a direct comparison between smooth and skeletal muscle myosin, the average force per cross-bridge was 0.8 and 0.2 pN, respectively. Surprisingly, smooth muscle myosin generates approximately 4 times greater average force per cross-bridge head than skeletal muscle myosin. Because average isometric force is the product of the cross-bridge unitary force and duty cycle, we are presently using a laser optical trap in an attempt to measure unitary events from single myosin molecules. This approach should allow us to determine whether an increase in unitary force, duty cycle, or both contribute to smooth muscle myosin's enhanced force-generating capacity compared with skeletal muscle myosin.
平滑肌每单位横截面积产生的力量与骨骼肌相同，但肌球蛋白含量仅为骨骼肌的五分之一。尽管这种明显的差异可以在组织或细胞水平上得到解释，但在分子水平上，平滑肌横桥产生的平均力量可能比骨骼肌横桥更大。为了验证这一假设，我们使用了一种体外运动分析方法（VanBuren等人，1994年），其中将鸡的硫代磷酸化砂囊平滑肌或胸肌骨骼肌单体肌球蛋白附着在硝酸纤维素表面。一条荧光标记的肌动蛋白丝连接到一个超顺应性（50 - 200纳米/皮牛）的玻璃微针上，使其与肌球蛋白表面接触。由肌球蛋白横桥拉动附着的肌动蛋白丝产生的等长力，根据校准微针的偏转程度来计算。通过测量附着在表面的肌球蛋白密度，我们估计了能够与接触肌球蛋白表面的一段肌动蛋白丝相互作用的肌球蛋白横桥数量。在平滑肌和骨骼肌肌球蛋白的直接比较中，每个横桥的平均力量分别为0.8皮牛和0.2皮牛。令人惊讶的是，平滑肌肌球蛋白每个横桥头产生的平均力量大约是骨骼肌肌球蛋白的4倍。由于平均等长力是横桥单位力和工作周期的乘积，我们目前正在使用激光光镊试图测量单个肌球蛋白分子的单位事件。这种方法应该能让我们确定与骨骼肌肌球蛋白相比，单位力的增加、工作周期的增加或两者都增加是否导致了平滑肌肌球蛋白增强的力量产生能力。
