School of Chemistry, Universidad Autónoma de Querétaro.
Department of Biochemical Engineering, Tecnológico Nacional de México/Instituto Tecnológico de Morelia.
J Vis Exp. 2024 Nov 8(213). doi: 10.3791/67186.
Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.
代谢主要由细胞能量可用性和环境条件协调。了解细胞如何适应不同营养和环境条件下的能量代谢的测定方法为阐明分子机制提供了有价值的信息。氧化磷酸化是大多数细胞中 ATP 的主要来源，线粒体呼吸活性是氧化磷酸化的关键组成部分，维持着用于 ATP 合成的线粒体膜电位。线粒体呼吸通常在缺少细胞环境的分离线粒体中进行研究。在这里，我们提出了一种在酵母完整细胞中定量测量线粒体呼吸的方法。该方法适用于任何酵母物种，尽管它通常用于酿酒酵母细胞。首先，测试酵母在特定条件下的生长情况。然后，将细胞用去离子水以 1:1 的比例（w/v）洗涤并重新悬浮。然后，将细胞置于带有恒速搅拌的氧测定仪室中，并使用克拉克电极来定量测量耗氧量。根据对电子传递链或 ATP 合成的影响，依次将一些分子放入室中并进行选择。首先加入 ATP 合酶抑制剂寡霉素来测量与 ATP 合成偶联的呼吸。然后，使用解偶联剂来测量最大呼吸能力。最后，加入一组电子传递链抑制剂以排除与线粒体呼吸无关的耗氧量。使用线性回归分析数据以获得斜率，代表耗氧率。该方法的优点是它是专门针对酵母线粒体呼吸的，保持了细胞环境的完整性。需要强调的是，用于线粒体呼吸定量的抑制剂在不同的酵母物种之间可能会有所不同。
