Schnyder T, Tittmann P, Winkler H, Gross H, Wallimann T
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
J Struct Biol. 1995 May-Jun;114(3):209-17. doi: 10.1006/jsbi.1995.1020.
Octamers of mitochondrial creatine kinase (Mi-CK) were modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryo-conditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view. Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the octamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis.
线粒体肌酸激酶（Mi-CK）的八聚体用硫醇特异性试剂N-乙基马来酰亚胺或金偶联衍生物马来酰亚胺基十一烷金进行修饰。结果表明，两种试剂对Mi-CK催化的抑制动力学相当，这表明大的金簇复合物可被反应性半胱氨酸接近。SDS-PAGE分析显示，每个Mi-CK单体的八个半胱氨酸中有两个被马来酰亚胺基十一烷金标记，对功能性马来酰亚胺基团具有相似的亲和力。标记分子的凝胶排阻色谱显示，硫醇修饰后Mi-CK的八聚体结构得以保留。在冷冻条件下通过电子显微镜观察的冻干金标记八聚体对比度增强，并且在端视图中显示出保存良好的四重对称性。金标记的Mi-CK的图像分析显示，平均端视图中有四个强对比度信号位于八聚体的周边，而分子中心仍然是电子半透明的。我们得出结论，每个单体被马来酰亚胺基十一烷金标记的两个半胱氨酸残基位于八聚体的周边，并且我们讨论了这些反应性半胱氨酸在酶催化中的可能作用。
