Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples
Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples
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DOI：
10.1016/j.cca.2006.05.034
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发表时间：
2006-12-01
影响因子：
5
通讯作者：
Rustin, Pierre
中科院分区：
文献类型：
--
作者：
Benit, Paule;Goncalves, Sergio;Rustin, Pierre
背景：当可用的生物材料数量有限时，组成呼吸链的五种复合物的活性测量已被证明是一个重大挑战。在这里，我们报告了一组三种方便的检测方法，可以在这种情况下进行这种测量。方法：一种测定依赖于顺序添加试剂来测量第一个复合物 IV 活性，然后测量复合物 II+III，然后测量 3-磷酸甘油脱氢酶 + 复合物 III 活性，最后测量分离的复合物 III 活性。第二次测定测量复合物 II 的活性，然后测量甘油-3-磷酸脱氢酶和异柠檬酸脱氢酶的活性。第三种测定方法测量鱼藤酮敏感复合物。 I 活性和随后的寡霉素敏感复合物 V 活性。结果：这些测定已成功用于少量显示呼吸链各种缺陷的人类细胞的提取物，以及视网膜和极早期小鼠胚胎的冷冻组织匀浆。结论：这组测定的优点在于其快速、简单的执行以及立即检测部分缺陷的能力，因为每个活性都可以与同一样品中测量的一个或两个其他活性进行比较。 (c) 2006 Elsevier B.V. 保留所有权利。
Background: The measurement of the activities of the five complexes comprising the respiratory chain has proven to be a major challenge when a limiting amount of biological material is available. Here we report a set of three convenient assays that allows this measurement under such circumstances. Methods: One assay relies on the sequential addition of reagents to measure first complex IV activity, followed by complex II+III, and then glycerol-3-phosphate dehydrogenase + complex III activities and finally isolated complex III activity. A second assay measures the activity of complex II followed by glycerol-3-phosphate dehydrogenase and isocitrate dehydrogenase. A third assay measures rotenone-sensitive complex. I activity and subsequently oligomycin-sensitive complex V activity. Results: These assays have been successfully used on extracts of small numbers of human cells displaying various defects in the respiratory chain, and on frozen tissue homogenates of retina and very early mouse embryos. Conclusions: The strength of this set of assays lies both in its rapid and simple execution and its capacity for immediate detection of partial defects, because each activity can be compared with one or two other activities measured in the same sample. (c) 2006 Elsevier B.V. All rights reserved.