Observation of two orientations from rigor cross-bridges in glycerinated muscle fibers.
Observation of two orientations from rigor cross-bridges in glycerinated muscle fibers.
复制标题
观察甘油肌纤维中严格的横桥的两个方向。
DOI：
10.1021/bi00368a055
复制
发表时间：
1986
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;Burghardt,TP
修订稿于 1986 年 4 月 29 日收到摘要：测量了专门附着在甘油肌纤维中肌球蛋白横桥的快速反应硫醇上的罗丹明标记的荧光偏振，以确定纤维不同生理状态下横桥的角度分布与温度的函数关系。为了研究低于 0 C 温度下的纤维，我们在沐浴液中添加了甘油作为防冻剂。我们发现，罗丹明探针的荧光偏振在 4℃ 下检测到等长活性、严密性、MgADP 和低离子强度松弛纤维中的交叉桥的不同角度分布。我们还发现，甘油存在下的严密性交叉桥可以相对于肌动蛋白丝保持至少两个不同的方向，一种在温度 T> 2°C 时占主导地位，另一种在温度 T<-10°C 时占主导地位。甘油的存在对于所研究的所有温度保持大致相同的方向。 T<-10°C 时的严格跨桥方向与甘油存在下的 MgADP 跨桥方向和 4°C 下的主动肌肉跨桥方向相似。这些发现表明，甘油存在下的严格跨桥在附着于肌动蛋白时至少有两个不同的方向：其中一个在高温下占主导地位，另一种在低温下或存在 MgADP 时占主导地位。后一种取向类似于等长活性纤维中存在的取向。这些发现表明，激活的跨桥循环中力的产生可能是由于这两个方向之间肌动蛋白附着的跨桥过渡而发生的。共价荧光探针（碘乙酰胺）四甲基罗丹明 (IATR) 特异性修饰甘油化兔腰肌纤维中肌球蛋白横桥的快速反应硫醇 (SH-1)（Borejdo 等人，1979），而不改变纤维产生钙激活张力的能力（Burghardt 等人，1983，1984）。通过检测作为激发光偏振函数的偏振荧光发射，我们可以获得探针角分布的独特特征，并从中推断出当肌纤维改变其生理状态时跨桥角分布的变化（Burghardt，1984）。我们之前曾报道过，在严格条件下，IATR 标记的交叉桥的探针角度分布与 MgADP 存在下的交叉桥的分布有明确的区别（Borejdo 等人，1982 年；Burghardt 等人，1983 年）。根据这些数据，我们得出结论，严格的跨桥保持与存在 MgADP 时不同的角度分布。这项工作是在室温下进行的。我们在这里报告了肌纤维中 IATR 标记的横桥上荧光偏振测量的数据，这些数据用于确定探针角度分布作为纤维各种生理状态温度的函数。测量结果是通过在含有 50% 甘油的溶液中孵育的纤维获得的，以防止纤维冻结。我们发现，在室温下，在严格加甘油的条件下，跨桥的探针角度分布与 MgADP 存在下的跨桥分布有明确的区别（在此温度下，MgADP 跨桥具有相同的探针角度分布。这项工作得到了美国公共卫生服务补助金 HL-16683 和肌肉萎缩症协会的支持。* 与作者在加州大学的通讯地址，旧金山。* 永久地址：Eótvós Loránd 生物化学系
Revised Manuscript Received April 29, 1986 abstract: The fluorescence polarization from rhodamine labels specifically attached to the fast-reacting thiol of the myosin cross-bridge in glycerinated muscle fibers has been measured todetermine the angular distribution of the cross-bridges in different physiological states of the fibers as a function of temperature. To investigate the fibers at temperatures below 0 C, we have added glycerol to the bathing solution as an antifreezing agent. We find that the fluorescence polarization from the rhodamine probe detects distinct angular distributions of the cross-bridges in isometric-active, rigor, MgADP, and low ionic strength relaxed fibers at 4 C. We also find that the rigor cross-bridges in the presence of glycerol can maintain atleast two distinct orientations relativeto the actin filament, one dominant at temperatures T> 2 C and another dominant at T<-10 C. MgADP cross-bridges in the presence of glycerol maintain approximately the same orientation for all temperatures investigated. The rigor cross-bridge orientation at T<-10 C is similar to both the MgADP cross-bridge orientation in the presence of glycerol and the active muscle cross-bridge orientation at 4 C. These findings show that the rigor cross-bridge in the presence of glycerol has at least two distinct orientations while attachedto actin: one of them dominant at high temperature, the other dominant at low temperature or when MgADP is present. The latter orientation resembles that present in isometric-active fibers. These findings suggest that force generation inthe activated cross-bridge cycle may occur as a result of an actin-attached cross-bridge transition between these two orientations. e covalent fluorescent probe (iodoacetamido) tetra-methylrhodamine (IATR) specifically modifies the fast-reacting thiol (SH-1) of the myosin cross-bridge in glycerinated rabbit psoas muscle fibers (Borejdo et al., 1979) without al-tering the fibers’ ability to produce calcium-activated tension (Burghardt et al., 1983, 1984). By detecting the polarized fluorescence emission as a function of the excitation light polarization, we can obtain distinctive features of the probe angular distribution and from them infer changes in the cross-bridge angular distribution when the muscle fiber changes itsphysiological state (Burghardt, 1984). We have reported previously that the probe angular dis-tribution for IATR-labeled cross-bridges in rigor is unam-biguously distinguishable from the distribution from cross-bridges in the presence of MgADP (Borejdo et al., 1982; Burghardt et al., 1983). From these data we concluded that the rigor cross-bridges maintain a different angular distribution from that maintained inthe presence of MgADP. This work was performed at room temperature. We report here data from fluorescence polarization mea-surements on IATR-labeled cross-bridges in muscle fibers that are used to determine the probe angular distribution as a function of temperature for various physiological states of the fiber. The measurements were obtained from fibers incubated in solutions with 50% glycerol to protect the fibers from freezing. Wefind that at room temperature the probe angular distribution for cross-bridges in rigor plus glycerol is unam-biguously distinguishable from that distribution for cross-bridges in the presence of MgADP (MgADP cross-bridges at this temperature have the same angular distribution of probes tThis work was supported by US Public Health Service Grant HL-16683 and by theMuscular Dystrophy Association.* Address correspondence to this author at the University ofCalifornia, San Francisco.* Permanent address: Department of Biochemistry, Eótvós Loránd