Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions

Abdulbaki Agbas, Partha Krishnamurthy, Mary L. Michaelis, Elias K. Michaelis
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引用次数: 9

Abstract

Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley & Sons, Inc.

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线粒体电子转移级联酶在培养神经元和特定脑区的活性评估
电子传递级联(ETC)酶活性及其动力学特征的测量对于评估健康和疾病或暴露于有毒物质后神经系统的线粒体功能非常重要。脑组织和神经元酶分析的优化是开发高通量分析格式的关键。本文描述了一个可靠的、可重复的评估线粒体ETC酶动力学(复合体I-IV)的逐步方案,这些线粒体来自不同大脑区域的少量组织,如海马、小脑和额叶皮层,或来自培养的神经元。详细介绍了从脑中分离细胞体和突触线粒体的差速离心和密度梯度离心方法,以及用分光光度法在微孔板或单比色皿中测定ETC活性的方法。介绍了易于遵循的分析布局和有用的提示,允许用户在3小时内执行这些分析。©2019 by John Wiley &儿子,Inc。
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