Ramanujan V K, Jo J A, Cantu G, Herman B A
Department of Cellular and Structural Biology, University of Texas Health Science, Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.
J Microsc. 2008 Jun;230(Pt 3):329-38. doi: 10.1111/j.1365-2818.2008.01991.x.
Traditional cuvette-based enzyme studies lack spatial information and do not allow real-time monitoring of the effects of modulating enzyme functions in vivo. In order to probe the realistic timescales of steric modifications in enzyme-substrate complexes and functional binding-unbinding kinetics in living cells without losing spatial information, it is imperative to develop sensitive imaging strategies that can report enzyme kinetics in real time over a wide dynamic range of timescales. Here we present a multi-photon excitation-based, ultra-fast photon detection using a streak camera and Laguerre expansion-based fast deconvolution approach for achieving high spatio-temporal resolution in monitoring real-time enzyme kinetics in single cells. In particular, we report spatially resolved, nanosecond-scale fluorescence dynamics associated with binding-unbinding kinetics of endogenous metabolic co-factor nicotinamide adenine dinucleotide with enzymes in intact living cells. By monitoring real-time kinetics of NAD(P)H-enzyme kinetics in primary hepatocytes isolated from young and aged mouse models, we observed that the mechanism of inhibition of mitochondrial respiration at complex I site is mediated by redistribution of free and protein-bound nicotinamide adenine dinucleotide pools and that this equilibrium redistribution is affected by age-related modifications in mitochondrial function. We describe unique advantages of Laguerre deconvolution algorithm in comparison with conventional lifetime analysis approaches. Non-invasive monitoring of metabolic dysfunctions in intact animal models is an attractive strategy for gaining insight into the dynamics of tissue metabolism in health and in various metabolic syndromes such as cancer, diabetes and aging-induced metabolic dysfunctions. Besides the example demonstrated above, we envisage that the proposed method can find applications in a variety of other situations where intensity-based approaches fall short owing to spectroscopic artefacts.
传统的基于比色皿的酶研究缺乏空间信息，且无法实时监测体内调节酶功能的效果。为了在不丢失空间信息的情况下探究酶 - 底物复合物中空间修饰的实际时间尺度以及活细胞中功能结合 - 解离动力学，开发能够在广泛的时间尺度动态范围内实时报告酶动力学的灵敏成像策略势在必行。在此，我们提出一种基于多光子激发的方法，利用条纹相机进行超快速光子检测，并采用基于拉盖尔展开的快速去卷积方法，以在监测单细胞实时酶动力学时实现高时空分辨率。特别是，我们报告了与完整活细胞中内源性代谢辅因子烟酰胺腺嘌呤二核苷酸与酶的结合 - 解离动力学相关的空间分辨纳秒级荧光动力学。通过监测从年轻和老年小鼠模型分离的原代肝细胞中NAD(P)H - 酶动力学的实时情况，我们观察到在复合体I位点抑制线粒体呼吸的机制是由游离和与蛋白质结合的烟酰胺腺嘌呤二核苷酸池的重新分布介导的，并且这种平衡重新分布受到与年龄相关的线粒体功能修饰的影响。我们描述了拉盖尔去卷积算法与传统寿命分析方法相比的独特优势。在完整动物模型中对代谢功能障碍进行非侵入性监测是一种有吸引力的策略，有助于深入了解健康状态以及癌症、糖尿病和衰老引起的代谢功能障碍等各种代谢综合征中组织代谢的动态变化。除了上述示例之外，我们设想所提出的方法可以在各种其他因光谱伪像导致基于强度的方法不足的情况下找到应用。
