Arata T
Department of Biology, Graduate School of Science, Osaka University, Japan.
Biochemistry. 1996 Dec 17;35(50):16061-8. doi: 10.1021/bi960803s.
It has been reported that chemically modified [with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS)] actin maintains its monomeric form and retains the ability to bind (and make chemical cross-links) to myosin head [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032; Arata, T. (1991) J. Biochem. (Tokyo) 109, 335-340]. Here, the interaction between MBS-G-actin and myosin subfragment 1 (S1) has been further studied by proteolytic susceptibility and chemical cross-linking. Two moles of MBS-actin monomers bound to 1 mol of myosin heads or S1 with different affinities. The first binding of MBS-G-actin to S1 strongly protected a 27-kDa/50-kDa junction of S1 heavy chain from trypsin digestion and also weakly protected a 50-kDa/20-kDa junction. The second binding protected a 50-kDa/20-kDa junction more strongly. ATP weakened these bindings more than 10-fold. MBS-G-actin was cross-linked to S1 by 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide, producing the 175-185-kDa doublet bands similar to those of F-actin and S1. The first binding produced a complex migrating at 175 kDa on gels [Hozumi, T. (1992) Biochemistry 31, 10071-10073] and the second binding further produced an 185-kDa complex, suggesting that two binding sites correspond to two spatially separated cross-linking sites. MBS-G-actin was also cross-linked by MBS to S1 when the first actin binds, producing only 180-kDa complex. In the presence of ATP or ADP, an 140-kDa complex was produced together with the 180-kDa complex, suggesting shifting of the binding site.
据报道，经间马来酰亚胺苯甲酸N-羟基琥珀酰亚胺酯（MBS）化学修饰的肌动蛋白保持其单体形式，并保留与肌球蛋白头部结合（并形成化学交联）的能力[贝塔切，N.，伯特兰，R.，&卡萨布，R.（1989年）《美国国家科学院院刊》86，6028 - 6032；荒田，T.（1991年）《生物化学杂志》（东京）109，335 - 340]。在此，通过蛋白水解敏感性和化学交联进一步研究了MBS - G -肌动蛋白与肌球蛋白亚片段1（S1）之间的相互作用。两摩尔的MBS -肌动蛋白单体以不同亲和力与1摩尔的肌球蛋白头部或S1结合。MBS - G -肌动蛋白与S1的首次结合强烈保护了S1重链的一个27 kDa / 50 kDa连接点免受胰蛋白酶消化，也微弱地保护了一个50 kDa / 20 kDa连接点。第二次结合对50 kDa / 20 kDa连接点的保护更强。ATP使这些结合减弱了10倍以上。MBS - G -肌动蛋白通过1 - 乙基 - 3 - [3 - （二甲基氨基）丙基] - 碳二亚胺与S1交联，产生了类似于F -肌动蛋白和S1的175 - 185 kDa双峰带。首次结合在凝胶上产生了一个在175 kDa处迁移的复合物[小泉，T.（1992年）《生物化学》31，10071 - 10073]，第二次结合进一步产生了一个185 kDa的复合物，表明两个结合位点对应于两个空间上分离的交联位点。当第一个肌动蛋白结合时，MBS - G -肌动蛋白也通过MBS与S1交联，仅产生180 kDa的复合物。在ATP或ADP存在下，与180 kDa复合物一起产生了一个140 kDa的复合物，表明结合位点发生了移动。
