Winkelmann D A, Kinose F, Chung A L
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
J Muscle Res Cell Motil. 1993 Aug;14(4):452-67. doi: 10.1007/BF00121297.
Conformational transitions in defined regions of the motor domain of skeletal muscle myosin involved in ATP hydrolysis, actin binding and motility were probed with monoclonal antibodies. Competition binding assays demonstrate that three different monoclonal antibodies react with spatially related sites on the myosin heavy chain. One recognizes a sequential epitope between residues 65 and 80 and has no effect on actin filament movement in an in vitro motility assay despite tight binding to myosin and acto-S1. The other two monoclonal antibodies react with sequential epitopes between residues 29 and 60 and both inhibit actin filament movement. A fourth monoclonal antibody reacts with the N-terminus of the heavy chain (residues 1-12) at a spatially distinct site on the myosin head and also inhibits actin filament movement. These four monoclonal antibodies have been mapped by immunoelectron microscopy to the large, actin binding region of the myosin head; however, the antibody binding sites remain accessible on rigor complexes of acto-S1. Thus, this group of monoclonal antibodies identify sequential epitopes in a mobile segment of the myosin heavy chain. In addition, two conformation-sensitive monoclonal antibodies are described that are affected by ATP and actin binding to myosin S1, and display distinct and marked inhibitory effects on actin filament movement. In contrast, an anti-light chain monoclonal antibody that binds near the myosin head-rod junction has little effect on the number and velocity of moving actin filaments. These results identify mobile regions on the myosin head that are perturbed by antibody binding and that may be linked to force production and motion.
利用单克隆抗体探究了骨骼肌肌球蛋白运动结构域特定区域中与ATP水解、肌动蛋白结合及运动性相关的构象转变。竞争结合试验表明，三种不同的单克隆抗体与肌球蛋白重链上空间相关的位点发生反应。一种识别65至80位残基之间的连续表位，尽管与肌球蛋白和肌动蛋白- S1紧密结合，但在体外运动试验中对肌动蛋白丝的运动没有影响。另外两种单克隆抗体与29至60位残基之间的连续表位发生反应，两者均抑制肌动蛋白丝的运动。第四种单克隆抗体在肌球蛋白头部空间上不同的位点与重链的N端（1至12位残基）发生反应，也抑制肌动蛋白丝的运动。通过免疫电子显微镜已将这四种单克隆抗体定位到肌球蛋白头部的大的肌动蛋白结合区域；然而，在肌动蛋白- S1的僵直复合物上抗体结合位点仍然可及。因此，这组单克隆抗体识别出肌球蛋白重链可移动片段中的连续表位。此外，还描述了两种构象敏感的单克隆抗体，它们受ATP和肌动蛋白与肌球蛋白S1结合的影响，并对肌动蛋白丝的运动表现出明显且显著的抑制作用。相比之下，一种结合在肌球蛋白头部-杆部连接处附近的抗轻链单克隆抗体对移动肌动蛋白丝的数量和速度影响很小。这些结果确定了肌球蛋白头部受抗体结合干扰且可能与力的产生和运动相关的可移动区域。
