Hiratsuka T
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
Biochemistry. 1998 May 19;37(20):7167-76. doi: 10.1021/bi973083d.
The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.
非共价荧光探针6-丙酰基-2-（二甲基氨基）萘（prodan）与肌球蛋白亚片段-1（S-1）以化学计量方式结合，且不影响S-1的ATP酶活性和肌动蛋白结合特性。ATP和肌动蛋白均不干扰prodan的结合。游离的prodan在520nm处呈现绿色发射峰。然而，与S-1结合的prodan以及S-1·ADP复合物分别在460nm和450nm处呈现蓝色发射峰，这使得能够轻松分离来自游离探针和结合探针的荧光贡献。在S-1·ADP·Pi状态下，蓝色发射峰进一步移至445nm，荧光增强幅度较大（4.5倍）。因此，在S-1存在的情况下，prodan仅在ATP水解过程中主要呈现蓝色荧光，从而持续可视化ATP酶反应。Mg2+、Ca2+和肌动蛋白激活的ATP酶稳态的初始速度可根据蓝色荧光变化方便地计算得出。不同核苷三磷酸（NTP）增强prodan蓝色荧光的能力顺序为ATP＞CTP＞UTP＞ITP＞GTP。该顺序与用原钒酸盐（Vi）捕获到S-1上的相应核苷二磷酸（NDP）核糖附近的疏水性程度顺序[平冢，T.（1984年）《生物化学杂志》（东京）96，155 - 162]以及不同NTP支持肌肉纤维中力产生的能力顺序[雷尼尔，M.等人（1993年）《生物物理杂志》64，A250]一致。相应的S-1·NDP·Vi复合物的形成速率也遵循此顺序，而NTP酶速率则遵循相反顺序。这些结果表明，核苷酸诱导的prodan荧光变化对应于核苷酸诱导的S-1构象状态。因此，在肌球蛋白ATP酶研究中使用prodan不仅为监测ATP酶反应，也为研究ATP水解过程中的结构变化提供了一种新的且有前景的方法。
