Arata T
Department of Biology, Faculty of Science, Osaka University.
J Biochem. 1991 Feb;109(2):335-40.
Polymerization of G-actin in the presence of salt and phalloidin was blocked by treatment of G-actin with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (designated as m-actin). The actin dimer produced by chemical crosslinking of F-actin with N,N'-p-phenylenedimaleimide did not polymerize and was still dimeric or tetrameric after further treatment with MBS (designated as d-actin). The m- and d-actins retained the ability to bind to myosin heads with apparent dissociation constants of 3-8 x 10(-6) and 3-5 x 10(-7) M, respectively. d-Actin formed a 1:1 actin monomer-myosin head complex. However, m-actin formed a 2:1 m-actin-head complex, suggesting the presence of at least two latent actin-binding sites on a myosin head. ATP weakens only 2- to 6-fold the binding of these complexes. One of two m-actins on a myosin head was replaced by d-actin. Native F-actin blocked the binding of both m- and d-actins to myosin heads in the presence and absence of ATP, although the affinities of myosin head for MBS-treated actins and F-actin are similar in the presence of ATP. These results suggest that there are at least three actin binding sites on a myosin head: one is responsible for binding of F-, m-, and d-actins, the second for binding of F- and m-actins, and the third for binding of F-actin at least in the presence of ATP. F-Actin binding to the third site may in some way block the first and second binding sites.
在盐和鬼笔环肽存在的情况下，用间马来酰亚胺苯甲酸N-羟基琥珀酰亚胺酯（MBS）处理G-肌动蛋白可阻断其聚合反应（称为m-肌动蛋白）。用N，N'-对苯二甲基马来酰亚胺对F-肌动蛋白进行化学交联产生的肌动蛋白二聚体不会聚合，在用MBS进一步处理后仍为二聚体或四聚体（称为d-肌动蛋白）。m-肌动蛋白和d-肌动蛋白保留了与肌球蛋白头部结合的能力，其表观解离常数分别为3 - 8×10⁻⁶和3 - 5×10⁻⁷ M。d-肌动蛋白形成1:1的肌动蛋白单体-肌球蛋白头部复合物。然而，m-肌动蛋白形成2:1的m-肌动蛋白-头部复合物，这表明在肌球蛋白头部至少存在两个潜在的肌动蛋白结合位点。ATP仅使这些复合物的结合减弱2至6倍。肌球蛋白头部上的两个m-肌动蛋白之一被d-肌动蛋白取代。在有和没有ATP的情况下，天然F-肌动蛋白均能阻断m-肌动蛋白和d-肌动蛋白与肌球蛋白头部的结合，尽管在有ATP的情况下肌球蛋白头部对MBS处理的肌动蛋白和F-肌动蛋白的亲和力相似。这些结果表明，肌球蛋白头部至少有三个肌动蛋白结合位点：一个负责结合F-、m-和d-肌动蛋白，第二个负责结合F-和m-肌动蛋白，第三个至少在有ATP的情况下负责结合F-肌动蛋白。F-肌动蛋白与第三个位点的结合可能以某种方式阻断第一和第二个结合位点。
