Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III CNIC;
Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III CNIC; Centro de Investigaciones Biomédica en Red de Fragilidad y Envejecimiento, Saludable-CIBERFES.
J Vis Exp. 2022 Jul 20(185). doi: 10.3791/63729.
Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na or K. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na environment of mitochondria.
泛醌 (CoQ) 在 线粒体内膜 (IMM) 中的池部分分段为依赖于复合体 I 或 FAD 的酶。这种细分可以通过使用 NADH 或琥珀酸作为电子供体在冷冻-解冻的线粒体中进行比较测定来轻松评估，其中测量细胞色素 c (cyt c) 的还原。该测定依赖于 Na 对 IMM 的影响，降低其流动性。在这里，我们提供了一种在存在 NaCl 或 KCl 的情况下测量 NADH-细胞色素 c 氧化还原酶活性和琥珀酸-细胞色素 c 氧化还原酶活性的方案。这些反应依赖于在比色杯中逐步混合试剂，在存在 Na 或 K 的情况下在 4 分钟内进行分光光度法测量。在存在特定酶抑制剂的情况下，同时进行相同的混合物，以减去吸光度的非特异性变化。NADH-细胞色素 c 氧化还原酶活性在存在这些阳离子中的任何一种时都不会降低。然而，琥珀酸-细胞色素 c 氧化还原酶活性在存在 NaCl 时降低。这个简单的实验突出了：1) Na 降低 IMM 流动性和 CoQ 转移的影响；2) 超复合体 I+III2 保护 CoQ 转移免受降低 IMM 流动性的影响；3) CI 和 CIII 之间的 CoQ 转移与 CII 和 CIII 之间的 CoQ 转移在功能上不同。这些事实支持 IMM 中存在功能分化的 CoQ 池的存在，并表明它们可以通过改变线粒体的 Na 环境来调节。
