The site and stoichiometry of the N-phenylmaleimide reaction with myosin when weakly-binding crossbridges are formed in skinned rabbit psoas fibers.
The site and stoichiometry of the N-phenylmaleimide reaction with myosin when weakly-binding crossbridges are formed in skinned rabbit psoas fibers.
复制标题
当带皮兔腰肌纤维中形成弱结合横桥时，N-苯基马来酰亚胺与肌球蛋白反应的位点和化学计量。
DOI：
10.1016/0005-2728(95)00094-6
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Schoenberg,M
中科院分区：
文献类型：
--
作者：
Ehrlich,A;Barnett,VA;Chen,HC;Schoenberg,M
用0.1 mM N-苯基马来酰亚胺（NPM）处理松弛的皮肤兔腰肌肌纤维1 h，将所有的横桥锁定在类似于肌球蛋白ATP横桥的弱结合状态。在这些条件下，NPM主要与肌球蛋白重链反应（巴内特等人（1992）Biophys. J. 61，358-367）。在这里，该反应的具体网站进行了探索。用Triton X-100处理小束兔腰大肌肌纤维，使纤维肌膜具有通透性。用0.1 mM [14 C]NPM处理束1 h，并均质化用于SDS-PAGE。43 ± 2.2%的肌纤维蛋白位于肌球蛋白重链带，与未处理的纤维相同。测定了每摩尔肌球蛋白重链2.2 ± 0.33摩尔NPM的烷基化化学计量。用胰蛋白酶消化，然后用二维薄层层析和反相高效液相色谱法，发现肌球蛋白重链上有两个主要位点用于NPM结合。该网站包含大约相同数量的连接NPM，这表明在研究的条件下，每个网站的反应化学计量是大约。1 mol NPM/mol肌球蛋白重链。将标记的胰蛋白酶肽与NPM反应的合成的SH 1和SH 2胰蛋白酶肽进行比较，并分析处理过的纤维束的ATP酶活性，表明肌球蛋白重链上的NPM反应位点是Cys-697（SH 2）和Cys-707（SH 1）。
Treatment of relaxed skinned rabbit psoas muscle fibers with 0.1 mM N-phenylmaleimide (NPM) for 1 h locks all of the crossbridges in a weakly-binding state resembling that of the myosin ATP crossbridge. Under these conditions, NPM reacts mainly with myosin heavy chain (Barnett et al. (1992) Biophys. J. 61, 358–367). Here the specific sites for that reaction are explored. Small bundles of rabbit psoas muscle fibers were treated with Triton X-100 to make the fiber sarcolemmas permeable. The bundles were treated with 0.1 mM [14C]NPM for 1 h, and homogenized for SDS-PAGE. 43 ± 2.2% of the muscle fiber protein ran in the myosin heavy chain band, the same as for untreated fibers. An alkylating stoichiometry of 2.2 ± 0.33 moles NPM per mole myosin heavy chain was determined. Exhaustive trypsin digestion followed by two-dimensional thin-layer chromatography and reverse-phase HPLC revealed two major sites on myosin heavy chain for NPM binding. The sites contained about the same amount of linked NPM, suggesting that the reaction stoichiometry of each site under the conditions studied is approx. 1 mol NPM/mol myosin heavy chain. Comparison of the labeled tryptic peptides with NPM-reacted synthetic SH1 and SH2 tryptic peptides and analysis of the treated fiber bundles' ATPase activity suggested that the sites for NPM reaction on myosin heavy chain when it locks crossbridges in a weakly-binding state are Cys-697 (SH2) and Cys-707 (SH1).