Salin Karine, Villasevil Eugenia M, Auer Sonya K, Anderson Graeme J, Selman Colin, Metcalfe Neil B, Chinopoulos Christos
Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow, UK
Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow, UK.
Physiol Rep. 2016 Oct;4(20). doi: 10.14814/phy2.13007. Epub 2016 Oct 24.
The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of "uncorrected" ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food-deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.
组织匀浆的使用极大地促进了线粒体功能的研究。然而，每消耗一个氧分子所产生的ATP量，即有效P/O比值，从未在组织匀浆中直接测量过。在此，我们结合并改进了先前用于通透细胞和分离线粒体的现有方法，以同时测量组织匀浆中的线粒体ATP生成量（JATP）和氧消耗量（JO）。与现有方法相比，一个主要的改进在于对否则会干扰ATP测定的ATP酶的控制：我们改进的技术有助于同时测量“未校正的”ATP合成速率和ATP水解速率，从而将所需的组织量和时间降至最低。最后，我们开发了一种计算有效P/O比值的新方法，该方法分别针对非线粒体ATP水解和呼吸速率对JATP和JO的测量进行校正。尽管匀浆2小时后活性下降，但褐鳟（Salmo trutta）肝脏匀浆中JATP和JO的测量具有高度可重复性。我们比较了喂食和禁食动物的线粒体特性，以证明该方法可以检测P/O比值中线粒体对营养状态的适应性。该方法简化了研究组织匀浆线粒体生物能量学的实验，无需差速离心或化学通透处理，也避免了使用非线粒体ATP酶抑制剂。我们得出结论，我们表征有效P/O比值的方法为在非常小的样本中研究线粒体功能开辟了新的可能性，而在这些样本中使用其他方法受到限制。
