Rimm D L, Kaiser D A, Bhandari D, Maupin P, Kiehart D P, Pollard T D
Department of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, Maryland 21205.
J Cell Biol. 1990 Dec;111(6 Pt 1):2405-16. doi: 10.1083/jcb.111.6.2405.
We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7-nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.
我们使用了一系列羧基末端缺失的重组肌球蛋白分子，以精确绘制22种单克隆抗体沿棘阿米巴肌球蛋白II尾部的结合位点。这些抗体结合到14个可区分的表位上，有些表位之间相隔不到10个氨基酸。通过电子显微镜观察到的结合位点的位置仅大致与这些位点在α-螺旋卷曲螺旋尾部上的物理位置相符。另一方面，表位图谱与竞争性结合研究精确相符：共享一个表位的所有抗体相互竞争与肌球蛋白的结合。具有相邻表位的抗体可以在长达5或6纳米的线性距离上相互竞争，许多相隔3 - 7纳米结合的抗体可以增强彼此与肌球蛋白的结合。大多数结合到尾部远端37纳米的抗体破坏八聚体微丝的组装，并根据结合位点的确切位置，在特定步骤阻止组装，产生例如单体、反平行二聚体、平行二聚体或反平行四聚体。这些抗体对组装的影响确定了微丝组装各个步骤所需的尾部位点。对截短的肌球蛋白II尾部组装的实验揭示了一组参与组装反应的互补位点（西纳德，J.H.，D.L.里姆，和T.D.波拉德。1990。《细胞生物学杂志》111:2417 - 2426）。结合到尾部远端但不影响组装的抗体似乎对肌球蛋白II亲和力较低。结合到尾部近端50纳米的抗体不抑制微丝的组装。许多结合到肌球蛋白II尾部的抗体，甚至一些对微丝组装没有明显影响的抗体，都可以抑制肌动球蛋白ATP酶活性以及粗提物中形成的肌动蛋白凝胶的收缩。一种结合在氨基酸1447和1467之间的抗体抑制1483位残基远端丝氨酸残基的磷酸化。
