...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >PRODUCTION AND PROPERTIES OF SKELETAL MYOSIN SUBFRAGMENT 1 SELECTIVELY LABELED WITH FLUORESCEIN AT LYSINE-553 PROXIMAL TO THE STRONG ACTIN-BINDING SITE
【24h】

PRODUCTION AND PROPERTIES OF SKELETAL MYOSIN SUBFRAGMENT 1 SELECTIVELY LABELED WITH FLUORESCEIN AT LYSINE-553 PROXIMAL TO THE STRONG ACTIN-BINDING SITE

机译：在强酪氨酸结合位点附近的赖氨酸553处选择性标记了荧光素的肌球蛋白亚片段1的产生和性质

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein, As a result, 0.9-1.0 mol of fluorescyl group/mol of S-1 was covalently incorporated exclusively into the 95 kDa heavy chain as monitored by spectroscopic measurements, The central 50 kDa segment included the main site of fluorescence attachment as assessed by gel electrophoresis. The extent of S-1-FHS conjugation is strongly sensitive to F-actin binding but not to the interaction of nucleotides. The formation of the rigor F-actin-S-1 complex decreased the level of S-1 labeling to 20% without any competition between actin and S-1 for FHS binding. The derivatization of S-1 did not alter the K+-ATPase activity, but it enhanced the Ca2+-ATPase and Mg2+-ATPase to 150% and 225%, respectively, whereas it lowered the actin-activated ATPase to only 75% of the original activity. A double-reciprocal plot of the ATPase rate against actin concentration indicated a 2-fold decrease of the V-max value for modified S-1, while the K-m for actin was unchanged. Cosedimentation experiments did not reveal disruption of the rigor acto-S-1 interaction by the bound fluorophore. The labeled S-1 heavy chain was isolated, and its total tryptic digest was fractionated by reverse-phase HPLC. Only two fluorescent peptides, designated P-1 and P-2, containing 15% and 85%, respectively, of the initial fluorescence were found, and after purification they were entirely sequenced. The major P-2 peptide spanned the heavy chain sequence Ala-545-Lys-561 with Lys-553 identified as the FHS-hyperreactive residue; the sequence of the minor P-1 peptide corresponded to Gly-638-Lys-641 with Lys-640 being linked to FHS. The location of Lys-553 in the S-1 primary structure is of particular interest as it is relevant to the primary stereospecific and hydrophobic actin-binding site thought to involve the helix(Gly-516-Phe-542)-loop(Pro-543-Thr-546)-helix(Asp-547-His-558) motif residing in the lower subdomain of the 50 kDa region. Lys-553 is positioned at the end of the latter helix, and the fluorescyl group bound to it may represent a valuable landmark to probe the functioning and orientational Properties of this strategic S-1 area during the acto-S-1-ATP interactions. [References: 18]
机译：我们首次描述了骨骼肌肌球蛋白亚片段1（S-1）与6- [荧光素-5（和6）-羧酰胺基]己酸（FHS）的琥珀酰亚胺酯的反应，该反应在pH 7.0下进行20分钟内，在15分钟内，存在比蛋白质多1.5-1.8倍摩尔过量的试剂。结果，将0.9-1.0 mol荧光基/ mol的S-1共价掺入到通过光谱测量监测到95 kDa重链。中心50 kDa片段包括通过凝胶电泳评估的荧光附着的主要位点。 S-1-FHS结合的程度对F-肌动蛋白的结合非常敏感，但对核苷酸的相互作用不敏感。严格的F-肌动蛋白-S-1复合物的形成将S-1标记的水平降低到20％，而肌动蛋白和S-1之间没有任何FHS结合竞争。 S-1的衍生化并没有改变K + -ATPase的活性，但是将Ca2 + -ATPase和Mg2 + -ATPase分别提高了150％和225％，而将肌动蛋白激活的ATPase降低到原始水平的75％。活动。 ATPase速率对肌动蛋白浓度的双倒数图表明，修饰的S-1的V-max值降低了2倍，而肌动蛋白的K-m不变。消沉实验未发现结合的荧光团破坏了严格的acto-S-1相互作用。分离出标记的S-1重链，并通过反相HPLC分离其总的胰蛋白酶消化物。仅发现了两种荧光肽，分别命名为P-1和P-2，分别含有15％和85％的初始荧光，纯化后对它们进行了完整测序。主要的P-2肽跨过重链序列Ala-545-Lys-561，其中Lys-553被鉴定为FHS高反应性残基。小P-1肽的序列对应于Gly-638-Lys-641，而Lys-640与FHS连接。 Lys-553在S-1一级结构中的位置特别受关注，因为它与一级立体特异性和疏水性肌动蛋白结合位点有关，认为该位点涉及螺旋（Gly-516-Phe-542）-环（Pro- 543-Thr-546）-螺旋（Asp-547-His-558）位于50 kDa区域的下部子域中。 Lys-553位于后一个螺旋的末端，与之结合的荧光基可能代表了一个有价值的地标，可用来探测在acto-S-1-ATP相互作用过程中此战略性S-1区域的功能和取向特性。 [参考：18]

著录项

相似文献

  • 外文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号