Neuromuscular Laboratory, Department of Neurosciences, University of Padova, Padova, Italy.
Nat Protoc. 2012 May 31;7(6):1235-46. doi: 10.1038/nprot.2012.058.
The assessment of mitochondrial respiratory chain (RC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RC enzymatic function (complexes I-IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.
评估线粒体呼吸链（RC）酶活性对于研究多种情况下的线粒体功能至关重要，包括线粒体疾病、糖尿病、癌症、衰老和神经退行性疾病，以及许多毒理学检测。肌肉是最常用的分析组织，因为它的代谢率高且易于获取，尽管其他组织和培养的细胞系也可以使用。我们描述了一种逐步方案，使用单波长分光光度计，可对来自各种物种和组织的少量肌肉、培养细胞和分离的线粒体的 RC 酶功能（复合物 I-IV）进行简单而可靠的评估。有效的组织破坏以及在每种测定中选择特定的缓冲液、底物、助剂和去污剂，使其浓度范围狭窄，可实现动力学的最大灵敏度、特异性和线性。此方案可在 3 小时内完成。
