Koffer A, Sleep J
MRC Cell Biophysics Unit, London, U.K.
J Muscle Res Cell Motil. 1987 Dec;8(6):541-7. doi: 10.1007/BF01567913.
Activation of skeletal muscle myosin and myosin subfragment-1 (S1) by actin purified from the cytoplasm of cultured BHK cells was studied using the fluorescence of pyrene-labelled BHK F-actin and its quenching by S1 and by an enzyme-linked ATPase assay. At non-saturating concentrations, both muscle and BHK actin activated skeletal muscle myosin to the same degree: at 30 degrees C and an ionic strength of 108 mM, 1 microM actin approximately doubled the ATPase of myosin or of S1. The association between BHK actin and S1 was also followed in a fluorescence stop flow: the rate of ATP binding monitored by the loss of light scattering upon dissociation of actin was again the same for BHK and muscle actin. The similarity of activation of myosin ATPase by BHK and muscle actin at low actin concentrations (i.e. the similarity of Vmax/Km) suggests that both Vmax and Km are similar for the two types of actin. The effect of varying filament length on actin activation of myosin ATPase was examined using pig plasma or BHK gelsolin to regulate the length. For both types of actin, maximum enhancement of the actomyosin ATPase activity was observed at an actin/gelsolin ratio of about 30:1, whereas inhibition was observed at lower ratios. Both activation and inhibition of actomyosin ATPase were apparent in the absence or presence of calcium; differences were observed only in the extent and the time course of the effect.
利用芘标记的BHK F-肌动蛋白的荧光及其被S1淬灭的特性，以及酶联ATP酶测定法，研究了从培养的BHK细胞胞质中纯化的肌动蛋白对骨骼肌肌球蛋白和肌球蛋白亚片段-1（S1）的激活作用。在非饱和浓度下，肌肉肌动蛋白和BHK肌动蛋白对骨骼肌肌球蛋白的激活程度相同：在30℃和离子强度为108 mM时，1 μM肌动蛋白可使肌球蛋白或S1的ATP酶活性增加约一倍。还通过荧光停流法追踪了BHK肌动蛋白与S1之间的结合：通过肌动蛋白解离时光散射的损失监测的ATP结合速率，BHK肌动蛋白和肌肉肌动蛋白再次相同。在低肌动蛋白浓度下（即Vmax/Km的相似性），BHK肌动蛋白和肌肉肌动蛋白对肌球蛋白ATP酶的激活相似，这表明两种类型的肌动蛋白的Vmax和Km都相似。使用猪血纤维蛋白原或BHK凝溶胶蛋白调节细丝长度，研究了细丝长度变化对肌动蛋白激活肌球蛋白ATP酶的影响。对于两种类型的肌动蛋白，在肌动蛋白/凝溶胶蛋白比例约为30:1时观察到肌动球蛋白ATP酶活性的最大增强，而在较低比例时观察到抑制作用。在有无钙的情况下，肌动球蛋白ATP酶的激活和抑制均很明显；仅在作用程度和时间进程上观察到差异。
