Hornikova Daniela, Herman Petr, Mejsnar Jiri, Vecer Jaroslav, Zurmanova Jitka
Faculty of Science, Charles University in Prague, Prague, Czech Republic.
Biochim Biophys Acta. 2009 Feb;1794(2):270-4. doi: 10.1016/j.bbapap.2008.10.018. Epub 2008 Nov 12.
Myofibrillar creatine kinase (CK) buffers the cellular ATP concentration during fluctuating ATP turnover in a muscle. In order to detect structural changes of the CK molecule due to bound substrates, the dynamics of free, ATP-bound, and ATP+creatine-bound CK were examined, using steady-state and time-resolved fluorescence spectroscopy. The intrinsic tryptophan fluorescence of non-labelled CK presented the smaller fluorescence lifetime 2.38 ns and rotation correlation time 27 ns for the CK-ATP (in comparison with the times 2.72 ns and 35 ns for the free CK), and their moderate return to the longer times 2.42 ns and 29 ns for the CK-ATP+creatine complex. Three conformations for the non-labelled CK were indicated also by different quenching of fluorescence by acrylamide. Data were confirmed by anisotropy experiments with CK-(FITC labelled), providing the same substrate dependence of the rotation times (34 ns, 27 ns and returning 30 ns). The results indicate the existence of three conformations arranged according to the "energy minimizing principle" by ligated substrates. In this way the data implicate another essential component of physiological control at the subcellular level in the transition of the nonreactive CK-ATP+creatine complex to the reactive enzyme molecule.
肌原纤维肌酸激酶（CK）在肌肉中ATP周转波动期间缓冲细胞内ATP浓度。为了检测由于结合底物导致的CK分子结构变化，使用稳态和时间分辨荧光光谱研究了游离的、ATP结合的以及ATP + 肌酸结合的CK的动力学。未标记的CK的内在色氨酸荧光显示，CK - ATP的荧光寿命较短，为2.38 ns，旋转相关时间为27 ns（与游离CK的2.72 ns和35 ns相比），而对于CK - ATP + 肌酸复合物，其适度恢复到较长的时间，分别为2.42 ns和29 ns。丙烯酰胺对荧光的不同猝灭也表明了未标记的CK存在三种构象。用CK - （FITC标记）进行的各向异性实验证实了这些数据，提供了相同的旋转时间对底物的依赖性（34 ns、27 ns和恢复到30 ns）。结果表明，通过连接底物，存在根据“能量最小化原则”排列的三种构象。通过这种方式，数据暗示了在亚细胞水平上，非反应性CK - ATP + 肌酸复合物向反应性酶分子转变过程中生理控制的另一个重要组成部分。
