Drew Barry, Leeuwenburgh Christiaan
Univ. of Florida, Biochemistry of Aging Laboratory, P.O. Box 118206, Gainesville, FL 32611, USA.
Am J Physiol Regul Integr Comp Physiol. 2003 Nov;285(5):R1259-67. doi: 10.1152/ajpregu.00264.2003. Epub 2003 Jul 10.
The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 microM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 microM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.
ATP的产生对于肌肉收缩、化学渗透稳态及正常细胞功能至关重要。许多研究测量了ATP含量或ATP产生过程中的定性变化，但很少有研究对分离线粒体中ATP的体内产生进行定量分析。鉴于了解线粒体能量能力在生物学、生理学、细胞功能障碍以及最终疾病病理和正常衰老方面的重要性，我们对一种市售的生物发光ATP测定法进行了改进，用于定量测量分离线粒体中ATP的含量和产生速率。生物发光测定法基于ATP与重组萤火虫荧光素酶及其底物荧光素的反应。对反应混合物以及相关ATP标准品的稳定性进行了定量分析。反应混合物和0.5微摩尔ATP标准品的发光信号分别以每分钟2.16%和1.39%的衰减率呈线性下降。对于25微摩尔的ATP标准品，由于与比尔-朗伯定律存在内在偏差，其发光信号呈对数衰减。此外，为了测试分离线粒体的功能，将它们与1毫摩尔和5毫摩尔的氧化磷酸化抑制剂寡霉素一起孵育。线粒体中ATP的产生速率分别下降了34%和83%。由于该测定法和方法的灵敏度及稳定性，我们能够在体内定量测量年龄和热量限制对年轻和老年Fischer-344大鼠脑和肝中分离线粒体的ATP含量及产生的影响。在这两种组织中，年龄和热量限制对ATP含量或ATP产生速率均无显著影响。本研究介绍了一种用于测量分离线粒体中ATP的高灵敏度、可重复且快速的方法。
