Hiratsuka T
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
Biophys J. 1997 Feb;72(2 Pt 1):843-9. doi: 10.1016/s0006-3495(97)78718-6.
A pyrene-labeled ATP (Pyr-ATP) in which a pyrene fluorophore is linked to the ribose moiety of ATP with a butyryl chain has been synthesized, together with the corresponding analog of ADP. The spectroscopic properties of two fluorescent analogs were found to be similar to those of 1-pyrenebutyric acid, making them photostable and highly sensitive probes for detecting changes in conformations around the nucleotide binding sites of proteins. Binding of Pyr-ADP to myosin subfragment-1 (S-1) resulted in a fluorescence quenching of about 70%. This binding was tight, with a dissociation constant (0.9 microM) similar to that of ADP itself. Formation of the stable ternary complex of Pyr-ADP with S-1 and orthovanadate could be monitored from the quench in pyrene fluorescence with a rate constant of 0.01 s-1. The final fluorescence intensity was about 20% of that for Pyr-ADP alone. Pyr-ATP was hydrolyzed by S-1 1.3 times faster than was ATP. Hydrolysis of Pyr-ATP was accompanied by an initial quenching of pyrene fluorescence with a subsequent recovery of the fluorescence. The fluorescence changes could be used to monitor the hydrolysis reaction continuously and measure the turnover rates of the analog. The fluorescence assay was sensitive, particularly under single turnover conditions, allowing hydrolysis reactions to be monitored at concentrations of S-1 and the analog as low as 50 nM.
已合成一种芘标记的ATP（Pyr-ATP），其中芘荧光团通过丁酰链与ATP的核糖部分相连，同时还合成了相应的ADP类似物。发现这两种荧光类似物的光谱性质与1-芘丁酸的光谱性质相似，这使得它们成为用于检测蛋白质核苷酸结合位点周围构象变化的光稳定且高度敏感的探针。Pyr-ADP与肌球蛋白亚片段-1（S-1）结合导致约70%的荧光猝灭。这种结合紧密，解离常数（0.9 microM）与ADP本身的解离常数相似。Pyr-ADP与S-1和原钒酸盐形成的稳定三元复合物可通过芘荧光猝灭进行监测，速率常数为0.01 s-1。最终荧光强度约为单独Pyr-ADP荧光强度的20%。Pyr-ATP被S-1水解的速度比ATP快1.3倍。Pyr-ATP的水解伴随着芘荧光的初始猝灭以及随后荧光的恢复。荧光变化可用于连续监测水解反应并测量类似物的周转速率。荧光测定法很灵敏，特别是在单周转条件下，能够在S-1和类似物浓度低至50 nM时监测水解反应。
