Kim Eldar, Bobkova Elena, Hegyi György, Muhlrad Andras, Reisler Emil
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Biochemistry. 2002 Jan 8;41(1):86-93. doi: 10.1021/bi0113824.
Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.
在体外运动性实验中，已表明ANP（N-(4-叠氮基-2-硝基苯基)腐胺）可使肌动蛋白丝在亚结构域2中的Gln-41与C末端的Cys-374之间发生链内交联，从而抑制与肌球蛋白产生力的作用[Kim等人（1998年）《生物化学》37卷，17801 - 17809页]。为了阐明这两个位点中哪一个的固定化会抑制肌动球蛋白马达，研究了具有部分重叠交联位点的肌动蛋白的特性。分别使用pPDM（N,N'-对苯二马来酰亚胺）和ABP [N-(4-叠氮苯甲酰基)腐胺]来获得在Cys-374与Lys-191（链间）以及Gln-41与Lys-113（链内）之间交联（约50%）的肌动蛋白丝。在体外运动性实验中，ANP、ABP和pPDM交联的丝在与肌球蛋白的滑动速度和产生力的方面表现出相似的抑制作用（约25%）。与肌动蛋白的ANP交联类似，pPDM和ABP交联不会改变S1与肌动蛋白的强结合以及肌动球蛋白ATP酶的V(max)和K(m)参数。这三种交联的相似作用揭示了亚结构域2/亚结构域1界面的结构元件之间的紧密耦合，并表明其动态灵活性对与肌球蛋白产生力的重要性。在停流实验中测试了肌动蛋白交联抑制肌球蛋白横桥循环中运动和产生力的限速步骤的可能性。对mantADP从肌动蛋白S1释放的速率以及ATP诱导的肌动蛋白S1解离的测量未揭示在这些复合物中未交联和ANP交联的肌动蛋白之间存在任何差异。根据交联的肌动蛋白丝中亚结构域2/亚结构域1界面受限的动态灵活性导致产生力与肌动球蛋白相互作用的其他方面之间的解偶联来讨论这些发现。
