Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1.
Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1.
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DOI：
10.1093/oxfordjournals.jbchem.a003181
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发表时间：
2002-06
影响因子：
2.7
通讯作者：
S. Maruta;Y. Mizukura;S. Chaen
中科院分区：
文献类型：
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作者：
S. Maruta;Y. Mizukura;S. Chaen
合成了一种新的荧光核糖修饰的三磷酸腺苷类似物2‘(3’)-O-[6-(N-(7-nitrobenz-2-oxa-1，3-diazol-4-yl)amino)hexanoic]-ATP，并研究了其与骨骼肌肌球蛋白亚段-1(S-1)的相互作用。S-1对NbD-ATP的水解率和二价阳离子依赖性与普通三磷酸腺苷相似。骨骼HMM支持NBD-ATP的肌动蛋白转位，其速度略高于常规ATP。在NBD-ATP中加入S1后，NBD荧光发生猝灭。在NBD-ATP完全水解为NBD-ADP后，荧光强度恢复。NBD-ATP荧光猝灭伴随着内源性色氨酸荧光的增强。这些结果表明，NBD-ATP荧光的猝灭反映了ATPase瞬态态的形成。通过跟踪NBD荧光的变化，监测了S-1.NBD-ADP.BeF(N)和S-1.NBD-ADP.AlF(-)配合物的形成。BEF(N)和AlF(4)(-)的屈服速率常数分别为7.38×10~(-2)S~(-1)和1.1×10~(-3)S~(-1)。这些值与本课题组以前报道的由S-1ADP.BeF(N)或AlF(4)(-)形成的Trp的本征荧光增强所估计的值相似。我们的新的ATP类似物似乎适用于肌球蛋白的动力学研究。
A new fluorescent ribose-modified ATP analogue, 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic]-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1.NBD-ADP.BeF(n) and S-1.NBD-ADP.AlF(4)(-) complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10(-2) s(-1) for BeF(n) and 1.1 x 10(-3) s(-1) for AlF(4)(-). These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1.ADP.BeF(n) or AlF(4)(-) reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.