Gregor M, Kubala M, Amler E, Mejsnar J
Department of Physiology and Developmental Biology, Faculty of Sciences, Charles University, Vinicná 7, CZ-128 44 Prague 2, Czech Republic.
Physiol Res. 2003;52(5):579-85.
Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity.
肌原纤维结合肌酸激酶EC 2.7.3.2（CK）是肌肉能量代谢的关键酶，已被选用于研究构成酶活性细胞控制基础的构象变化。为了进行荧光光谱测量，CK分子用IAF（5-碘乙酰氨基荧光素）和ErITC（异硫氰酸5'-赤藓红）进行了双标记。通过测量从荧光素到赤藓红的荧光共振能量转移（FRET）来获取有关供体-受体对距离的信息。频域寿命测量评估天然CK分子中的供体-受体距离为7.8 nm。Förster半径等于5.3 nm，分辨率范围为0.2至1.0 nm。通过高盐浓度处理测试了赤藓红-荧光素标记（EFL）对CK分子人工构象变化的影响。由His-97和Cys-283定义并从三维模型得出的转变距离，对于CK分子的开放（无活性）形式为0.766 nm，对于封闭（反应性）形式为0.277 nm。这样，考虑到0.489 nm的差异，所用光谱方法的分辨率范围很显著。然而，通过己糖激酶偶联测定法评估的CK酶活性降至天然酶活性的1%。EFL适用于描述肌酸激酶调节所暗示的构象行为。然而，观察到的抑制作用将EFL限制在天然催化活性期间构象变化的研究中。
