Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE.
Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE.
复制标题
(碘乙酰胺基)丙烯自旋标记肌球蛋白亚片段 1 装饰肌纤维的特异性和方向：使用 SDS-PAGE 定位蛋白质结合自旋标签。
DOI：
10.1021/bi00485a023
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Burghardt,TP
中科院分区：
文献类型：
--
作者：
Ajtai,K;Pótó,L;Burghardt,TP
梅奥基金会生物化学与分子生物学系，明尼苏达州罗切斯特55905摘要：硝基自旋标记（iodoacetamido） proxyl （IPSL）特异性地、刚性地附着在肌球蛋白亚片段1 （SI）上的巯基1 （SHI）上。使用电子顺磁共振（EPR）技术将自旋标签定位在肌球蛋白电泳分离的蛋白水解片段上，证明了该标签对SHI的特异性。对探针在SHI上的刚性研究表明，在存在或不存在MgADP或Mg ATP的情况下，IPSL可以固定在SI表面。经IPSL-S1修饰的肌纤维的EPR谱显示，IPSL-S1在与MgADP结合后从其方向进行了严格的旋转。由核苷酸结合引起的角位移比用（雄性）节奏自旋标签检测到的要大[Ajtai， K., French, a.r., & Burghardt, t.p. (1989) Biophys]。[j] . 56,535 -541]，证明IPSL在肌球蛋白桥上定向，有利于检测在向MgADP状态转变过程中的桥旋转。
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905 Received January 17, 1990; Revised Manuscript Received April 12, 1990 abstract: The nitroxide spin label (iodoacetamido) proxyl (IPSL) was specifically and rigidly attached to sulfhydryl 1 (SHI) on myosin subfragment 1 (SI). The specificity of this label for SHI was demonstrated by using a technique where the spin label is localized on the electrophoresis-isolated proteolytic fragments of myosin using electron paramagnetic resonance (EPR). Studies of the rigidity of the probe on SHI indicate that the IPSL is immobilized on the surface of SI in the presence and absence of the nucleotides MgADP or Mg ATP. The EPR spectrum of muscle fibers decorated with IPSL-S1 shows that the IPSL-S1 rotates from its orientation in rigor upon binding MgADP. The angular displacement due to nucleotide binding is larger than that detected with the (maleimido) tempo spin label [Ajtai, K., French, A. R., & Burghardt, T. P.(1989) Biophys. J. 56, 535-541], demonstrating that the IPSL is oriented on the myosin cross-bridge in a manner that is favorable for detecting cross-bridge rotation during the rigor to MgADP state transition.