Kim E, Phillips M, Hegyi G, Muhlrad A, Reisler E
Department of Chemistry and Biochemistry, Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
Biochemistry. 1998 Dec 22;37(51):17793-800. doi: 10.1021/bi9812874.
Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1(A1) and S1(A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.
在长间距螺旋中相邻单体上的Gln - 41和Cys - 374之间与ANP（N -（4 - 叠氮基 - 2 - 硝基苯基） - 腐胺）部分交联的肌动蛋白丝被解聚，并分级分离成长itudinal交联二聚体（s(o)20,w = 5.55 +/- 0.22 S）、三聚体（s(o)20,w = 6.93 +/- 0.12 S）和高阶寡聚体池。在芘基G - 肌动蛋白存在下，肌球蛋白亚片段（S1）与交联二聚体的竞争结合实验表明，第一个S1分子与肌动蛋白二聚体的结合比第二个S1分子强约2个数量级。在类似条件下，未聚合的交联肌动蛋白物种激活S1的MgATPase的程度仅为F - 肌动蛋白激活程度的几倍，后者可激活70倍。交联二聚体、三聚体和寡聚体通过MgCl2聚合成丝的速度比未交联的肌动蛋白快。在电子显微镜照片中，这些丝有时看起来更短，并且比未交联的肌动蛋白丝更易弯曲。少量交联肌动蛋白二聚体引发了S1诱导的肌动蛋白聚合，但对于纯群体的交联二聚体、三聚体和寡聚体，S1的聚合作用受到抑制。交联二聚体并未减小S1同工酶S1(A1)和S1(A2)引发的肌动蛋白聚合之间的动力学差异。根据电子显微镜证据，由S1聚合的交联肌动蛋白寡聚体产生的箭头状结构比未交联的肌动蛋白短得多。这些结果表明肌动蛋白 - 肌动蛋白侧向相互作用对于肌球蛋白ATPase的激活以及S1引发的肌动蛋白聚合的重要性。
