FLUOROMETRIC ANALYSIS-ISOZYMIC FORMS OF CREATINE KINASE

拟议的调查旨在应用相位荧光分析， 蛋白质三级和四级结构的相互依赖性， 肌酸激酶的二聚体同工酶。 这个根本问题 处理的是亚基折叠和 非变构的整体构象中的亚基组织， 多聚体蛋白 荧光分析的最新进展提供 敏感和搜索的定量标准，以比较具体的 结构成分，包括位点间距离，蛋白质旋转 速率，不对称的亚单位组织和运动的自由， 活动站点域。 亚基折叠和亚基缔合的动力学 将通过连续测量共振能量转移来分析 效率 寿命测量和时间分辨各向异性衰减 相位/调制荧光法，构成基本的分析程序。 总之，这些程序构成了一种实验方法， 评价三级和四级结构在生物化学中的贡献 三维形态的形成和维持， 肌酸激酶与底物和底物类似物相互作用。 而 用作非变构寡聚体中亚基相互作用的模型 肌酸激酶具有几个有趣的特征，是一种 重要的酶，因为它在神经肌肉中的中心作用 能量代谢 该调查代表了 更好地理解亚基和寡聚体结构的相互依赖性 在非变构催化、调节、区室化和稳定化中 对生物功能至关重要。

The proposed investigation seeks to apply phase fluorometry to an analysis of the interdependency of the tertiary and quaternary protein structure of the dimeric isozymes of creatine kinase. The fundamental question addressed is the nature of the interactions between subunit folding and subunit organization in the overall conformation of non-allosteric, multimeric proteins. Recent advances in fluorometric analysis provide sensitive and searching quantitative criteria by which to compare specific structural components including site-site distances, protein rotational rates, asymmetric subunit organization and motional freedom within the active site domain. Kinetics of subunit folding and subunit association will be analyzed by continuous measurement of resonance energy transfer efficiency. Lifetime measurements and time resolved anisotropy decay using phase/modulation fluorometry, constitute essential analytical procedures. Taken together, the procedures constitute an experimental approach to evaluating the contribution of tertiary and quaternary structure in the formation and maintenance of three-dimension conformation and response to interaction with substrate and substrate analog by creatine kinase. While used as a model for subunit interactions in non-allosteric oligomeric enzymes, creatine kinase exhibits several interesting features and is an important enzyme to study because of its central role in neuromuscular energy metabolism. The inquiry is representative the long term goal of better understanding the interdependency of subunit and oligomer structure in non-allosteric catalysis, regulation, compartmentation and stabilization essential for biological function.