Highsmith S, Eden D
Biochemistry. 1985 Aug 27;24(18):4917-24. doi: 10.1021/bi00339a029.
Heavy meromyosin (HMM) and myosin subfragment 1 (S1) were prepared from myosin by using low concentrations of alpha-chymotrypsin. The light chain distribution in HMM was identical with that of myosin, within experimental error, when analyzed on 12% polyacrylamide gels after electrophoresis. Specific birefringences and birefringence decay times were measured by transient electrical birefringence in 5 mM KCl, 5 mM tris(hydroxymethyl)aminomethane (pH 7), and 1 mM MgCl2 at 4 degrees C under gentle conditions that reduced the CaATPase activity by less than 10%. For solutions of HMM, by use of electric field pulses shorter than 0.5 microseconds, the birefringence decay signal from the S1 portions of HMM could be resolved and the rotational motions of the S1 moieties observed directly. The rotation relaxation time, adjusted to 20 degrees C, was 0.34 microseconds; this is in quantitative agreement with previous hydrodynamic results obtained by using covalently attached probes. The assignment of the fast decay time obtained with HMM to the S1 portions was confirmed by birefringence decay measurements on free S1, for which the relaxation time was 0.13 microseconds, corrected to 20 degrees C. The specific birefringences for S1 and HMM, respectively, were 0.37 X 10(-6) and 12.8 X 10(-6) (cm/statvolt)2. Thus, for much longer electric field pulses, the signal from HMM is due almost entirely to its subfragment 2 (S2) portion, and its rotational dynamics can also be monitored directly by using electrical birefringence. The decay of the signal from the S2 portion could be adequately fit without evoking bending of the S2 portion of HMM other than at its junction with S1.
通过使用低浓度的α-胰凝乳蛋白酶从肌球蛋白制备重酶解肌球蛋白（HMM）和肌球蛋白亚片段1（S1）。当在12%聚丙烯酰胺凝胶上进行电泳分析后，在实验误差范围内，HMM中的轻链分布与肌球蛋白的相同。在4℃下，于5 mM氯化钾、5 mM三（羟甲基）氨基甲烷（pH 7）和1 mM氯化镁中，通过瞬态电双折射在温和条件下测量比双折射和双折射衰减时间，该条件使CaATP酶活性降低不到10%。对于HMM溶液，通过使用短于0.5微秒的电场脉冲，可以分辨出来自HMM的S1部分的双折射衰减信号，并直接观察到S1部分的旋转运动。调整到20℃时，旋转弛豫时间为0.34微秒；这与先前使用共价连接探针获得的流体动力学结果在定量上一致。通过对游离S1的双折射衰减测量证实了将HMM获得的快速衰减时间归属于S1部分，其弛豫时间校正到20℃时为0.13微秒。S1和HMM的比双折射分别为0.37×10⁻⁶和12.8×10⁻⁶（厘米/静电伏特）²。因此，对于长得多的电场脉冲，来自HMM的信号几乎完全归因于其亚片段2（S2）部分，并且其旋转动力学也可以通过使用电双折射直接监测。除了在其与S1的连接处外，无需引发HMM的S2部分弯曲就可以充分拟合来自S2部分的信号衰减。
