Manuck B A, Seidel J C, Gergely J
Biophys J. 1986 Aug;50(2):221-30. doi: 10.1016/S0006-3495(86)83456-7.
The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.
在有和没有二磷酸腺苷或5'-腺苷酰-亚氨基二磷酸（AMPPNP）存在的情况下，研究了肌动蛋白与自旋标记的重酶解肌球蛋白（MSL-HMM）的相互作用，以确定单头结合和双头结合的作用。通过比较分别由饱和转移电子顺磁共振（ST-EPR）和沉降法测定的固定化头部的分数（fi）与结合分子的分数（fs），推断出单头与肌动蛋白结合的程度。ST-EPR测量取决于自旋标记物在HMM头部与肌动蛋白相互作用时与HMM头部刚性结合的运动减少。在用核苷酸滴定肌动蛋白-MSL-HMM的过程中，我们测量了由于MSL-HMM从肌动蛋白上解离而导致的fi和fs的变化。用ADP滴定，fs变化很小，保持在0.8以上，而在10mM ADP时fi降至约0.5，这一结果与MSL-HMM与肌动蛋白的广泛单头结合一致。用AMPPNP滴定，未检测到单头结合；即，fi和fs平行下降。无需假设结合头部的核苷酸诱导状态，其运动特性与僵直头部不同，就能解释这些结果。
