LOCALIZATION OF EPITOPES AND FUNCTIONAL-EFFECTS OF 2 NOVEL MONOCLONAL-ANTIBODIES AGAINST SKELETAL-MUSCLE MYOSIN
LOCALIZATION OF EPITOPES AND FUNCTIONAL-EFFECTS OF 2 NOVEL MONOCLONAL-ANTIBODIES AGAINST SKELETAL-MUSCLE MYOSIN
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DOI：
10.1007/bf01843575
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发表时间：
1990-06-01
影响因子：
2.7
通讯作者：
MUHLRAD, A
中科院分区：
文献类型：
--
作者：
DANGOOR, M;SILBERSTEIN, L;MUHLRAD, A
用两种抗人骨骼肌肌球蛋白的单克隆抗体研究了兔骨骼肌肌球蛋白S-1的功能与一级和三级结构之间的关系。S-1的重链被胰蛋白酶切割成27 kDa、50 kDa和20 kDa的三个片段，从N-末端按此顺序排列。第一抗体的表位被指定为S-1的N-末端1-23个氨基酸段，因为它与S-1的27 kDa N-末端胰蛋白酶片段反应，但不与缺少上述氨基酸段的27 kDa片段的衍生物反应。第二抗体的表位被分配到S-1的中心50 kDa结构域的3 kDa N-末端区域。该分配基于S-1的蛋白水解和光化学裂解以及通过特异性抗体标记其N-末端。在抗体与肌球蛋白的旋转阴影复合物的电子显微照片上，抗体与肌球蛋白头部结合。在显微照片上的测量表明，肌球蛋白的头-尾连接与“抗27 K”和“抗50 K”表位之间的距离分别为14 nm和17 nm。两种抗体对S-1都具有高亲和力。抗50 K抗体与S-1的亲和力在肌动蛋白结合后降低，而抗27 K抗体的亲和力不受S-1与F-肌动蛋白结合的影响。“抗50 K”抗体抑制S-1的K+（EDTA）和肌动蛋白激活的ATP酶活性，而“抗27 K”抗体则无此作用。结果表明，要么“抗-50 K”的表位靠近肌动蛋白或S-1的ATP结合位点，要么这些位点与表位之间存在通信，表达为传播的构象变化。
Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeltal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin.sbd.27 kDa, 50 kDa and 20 kDa.sbd.aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the ''anti-27 K'' and ''anti-50 K'' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the ''anti-50 K'' to S-1 decreased upon actin binding, while that of the ''anti-27 K'' was not affected by binding of S-1 to F-actin. The ''anti-50 K'' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the ''anti-27 K'' had no effect. The results indicate that either the epitope of the ''anti-50 K'' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.