Rotational dynamics of actin-bound myosin heads in active myofibrils.
Rotational dynamics of actin-bound myosin heads in active myofibrils.
复制标题
活跃肌原纤维中肌动蛋白结合的肌球蛋白头的旋转动力学。
DOI：
10.1021/bi00065a038
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Thomas,DD
中科院分区：
文献类型：
--
作者：
Berger,CL;Thomas,DD
摘要：我们用饱和转移电子顺磁共振(ST-EPR)测量了肌原纤维中肌动蛋白结合的肌球蛋白头的亚毫秒旋转运动。用马来酰亚胺氮氧化物衍生物(MSL)自旋标记交叉桥，先前已证明在低离子强度下稳态ATPase活性时，这种衍生物在溶液中肌动蛋白结合的肌球蛋白头上经历微秒旋转运动[Berger，C.L.，Svensson，E.C.，&Thomas，DD()过程。娜塔莉。阿卡德。SCI。美国85,8573]。为了确定这是否也适用于生理缓冲条件下的肌原纤维晶格中的交叉桥，我们在自旋标记的肌原纤维悬浮液中光解笼中的ATP后的短暂稳定期间进行了ST-EPR实验。肌原纤维被EDC[L-乙基-3-[3-(二甲氨基)丙基]碳二亚胺]部分交联，以防止其在激活时缩短。在生理离子强度下，使用肌球蛋白结合分析的蛋白分解速率[Duong，AM，&Reisler，E.(1989)BioChemical 28,3502]，以生化方法测定活性肌原纤维中肌球蛋白头的比例，然后这些数据被用来校正活性肌原纤维的ST-EPR谱，以确定是否存在独立的肌球蛋白头，这些头被假设经历与松弛时相同的运动。在生理离子强度下(µ=165 mm)，肌球蛋白结合的肌球蛋白头在激活的肌原纤维中有相当大的微秒旋转运动(rr=3.5±1.1µ$)。对于低离子强度(µ=45 mm)的活性肌原纤维，也获得了类似的结果(=3.2±0.8微米)，证实了在溶液中所做的工作。因此，在生理条件下，甚至在肌原纤维晶格的限制下，主动循环的肌动蛋白附着的肌球蛋白头部在微秒时间尺度上旋转移动。由于部分EDC固定的肌原纤维是溶液中等长收缩肌纤维的极佳模拟，这些微秒级的旋转运动很可能与活体肌肉收缩的分子机制直接相关。
Revised Manuscript Received February 1, 1993 abstract: We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, DD (1989) Proc. Natl. Acad. Sci. USA 85, 8573]. To determine whether this is also truefor cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performedST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [l-ethyl-3-[3-(dimethylamino) propyl] carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, AM, & Reisler, E.(1989) Biochemistry 28, 3502], These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (µ= 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (rr= 3.5±1.1 µ $) in the active myofibrils. Similar results (,= 3.2±0.8 µ $) were obtained with active myofibrils at low ionic strength (µ= 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.