Sowerby A J, Seehra C K, Lee M, Bagshaw C R
Department of Biochemistry, University of Leicester, U.K.
J Mol Biol. 1993 Nov 5;234(1):114-23. doi: 10.1006/jmbi.1993.1567.
Recent developments of in vitro motility assays have allowed the sliding velocity and force generation to be measured when a single actin filament interacts with a small number of immobilized myosin molecules. In contrast, the associated ATPase activities have been estimated from the whole population of molecules in the flow cell using steady-state kinetics. For a more unambiguous estimate of the crossbridge step size, it would be desirable to measure the ATPase activity of those molecules actually under observation in the in vitro assay in real time. As a start to solving this formidable problem we have investigated the use of fluorescent ATP analogues as probes to measure the ATPase activity of immobilized myosin filaments. Turnover rates for the substrate analogue, FEDA-ATP (an analogue in which a fluorescein moiety is linked via an ethylenediamine chain to the ribose of ATP) were recorded by displacement of the steady-state intermediate with excess ATP. Using epifluorescence light microscopy, small clumps of rabbit skeletal and scallop striated muscle synthetic thick filaments and single native clam (Mercenaria) red adductor muscle thick filaments were assayed. The latter filaments contain several thousand myosin molecules and thus they represent the application of transient kinetic methodology on the zeptomole scale. In the presence of Ca2+, the derived rate constants for FEDA-ATP turnover are close to those expected for the same preparations in solution (0.06 s-1 for rabbit skeletal, 0.2 s-1 for scallop and clam adductor muscle myosins), indicating that immobilization does not have a significant effect on the ATPase activity. In the absence of Ca2+, molluscan preparations show a slower FEDA-ATP turnover rate but they do not appear as well regulated as in solution. In such microscope assays the observed displacement rate does not reflect the true turnover rate owing to photobleaching, and possibly regulatory light chain depletion. Future developments for extending this assay to the actin-activated state are discussed.
近期体外运动分析技术的发展，使得在单根肌动蛋白丝与少量固定化肌球蛋白分子相互作用时，能够测量其滑动速度和产生的力。相比之下，相关的ATP酶活性是通过稳态动力学，从流动池中所有分子的总体来估算的。为了更明确地估计横桥步长，希望实时测量体外分析中实际观察到的那些分子的ATP酶活性。作为解决这一艰巨问题的开端，我们研究了使用荧光ATP类似物作为探针，来测量固定化肌球蛋白丝的ATP酶活性。通过用过量ATP取代稳态中间体，记录底物类似物FEDA-ATP（一种荧光素部分通过乙二胺链连接到ATP核糖上的类似物）的周转速率。使用落射荧光显微镜，对兔骨骼肌和扇贝横纹肌的合成粗丝小团块以及单个天然蛤（美洲帘蛤）红色内收肌粗丝进行了分析。后者的粗丝包含数千个肌球蛋白分子，因此它们代表了zeptomole尺度上瞬态动力学方法的应用。在Ca2+存在的情况下，FEDA-ATP周转的推导速率常数接近溶液中相同制剂预期的值（兔骨骼肌为0.06 s-1，扇贝和蛤内收肌肌球蛋白为0.2 s-1），这表明固定化对ATP酶活性没有显著影响。在没有Ca2+的情况下，软体动物制剂显示出较慢的FEDA-ATP周转速率，但它们似乎不像在溶液中那样受到良好的调节。在这种显微镜分析中，由于光漂白以及可能的调节轻链耗竭，观察到的位移速率并不反映真正的周转速率。讨论了将该分析扩展到肌动蛋白激活状态的未来发展。
