Mihashi K, Ooi A, Hiratsuka T
Department of Physics, Faculty of Science, Nagoya University, Aichi.
J Biochem. 1990 Mar;107(3):464-9. doi: 10.1093/oxfordjournals.jbchem.a123068.
In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.
为了研究由肌球蛋白亚片段 -1（S1）、ADP 和原钒酸盐（Vi）组成的三元复合物，即 S1·ADP·Vi 的灵活性，对结合的 ADP 的交换性进行了研究。通过凝胶过滤分离出 S1·ADP·Vi 三元复合物后，在 0.5℃下加入 ADP 的荧光类似物 3'-O-(N-甲基邻氨基苯甲酰基)-ADP（Mant-ADP）。添加的 Mant-ADP 通过取代结合的 ADP 非常缓慢地掺入三元复合物中。核苷酸交换发生时，S1 的 ATP 酶活性未恢复。同样，根据平冢（3,4）的方法通过凝胶过滤制备并分离的 S1·Mant-ADP·Vi 三元复合物，在 4.5℃下与 ADP（2.4 mM）孵育。S1·Mant-ADP·Vi 与 ADP 的核苷酸交换分两个阶段进行，表观速率分别为 4.5×10⁻⁴ s⁻¹（快速阶段）和 6.7×10⁻⁶ s⁻¹（缓慢阶段）。在 S1(A1)同工酶中也观察到结合核苷酸的双相交换，这表明双相交换并不对应于两种 S1 同工酶。快速和缓慢阶段的表观速率随着添加的 ADP 浓度增加而增加，但在 ADP 浓度约为 2 mM 时达到饱和，这表明核苷酸交换反应涉及一个（或多个）对溶液中游离 ADP 浓度不敏感的步骤。这个步骤可能是一个可逆异构化。
