Dan-Goor M, Kessel M, Muhlrad A
Hebrew University-Hadassah School of Dental Medicine, Department of Oral Biology, Jerusalem, Israel.
Biochim Biophys Acta. 1990 Apr 19;1038(2):269-73. doi: 10.1016/0167-4838(90)90215-2.
Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.
肌球蛋白含有反应性赖氨酸残基，其被2,4,6-三硝基苯磺酸盐三硝基苯化的速度比其他赖氨酸快得多。在这里，我们借助抗三硝基苯基抗体找到了这些残基在肌球蛋白一级结构和空间结构中的位置。这种抗体是在兔子体内针对三硝基苯基血蓝蛋白产生的。它与三硝基苯化的肌球蛋白以及三硝基苯化肌球蛋白的一些胰蛋白酶片段发生反应。通过对蛋白质免疫印迹反应进行分析，发现该抗体优先与肌球蛋白头部27 kDa的N端片段发生反应，而与肌球蛋白杆状部分的轻酶解肌球蛋白区域的反应较弱。27 kDa片段含有反应性最强的赖氨酸残基，而中等反应性的赖氨酸残基位于轻酶解肌球蛋白区域。在旋转阴影的三硝基苯化肌球蛋白-抗体复合物的电子显微镜图像上观察到了抗体表位的位置。对于头部的表位（反应性赖氨酸残基）和杆状部分的表位（中间反应性赖氨酸残基），其与肌球蛋白头部-杆状连接点的距离分别测定为13 nm和113 nm。
