Current Protocols in Toxicology
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.
brain subcellular fractionation, electron transport chain enzymes, mitochondria, neuroblastoma cultures, primary neuronal cultures
Agbas A, Krishnamurthy P, Michaelis ML, Michaelis EK. Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions. Current Protocols in Toxicology. 2019; 80(1). doi: 10.1002/cptx.73.