Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis.
Mitochondrial dysfunction and degeneration are associated with many neurodegenerative disorders. A dysfunctional mitochondrial electron transport chain (ETC) impairs ATP production and accelerates the generation of free radicals. To evaluate mitochondrial function, reliable methods are needed. Conventional spectrophotometric assays may not eliminate interference from nonspecific enzyme activities and do not measure quantities of specific ETC complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to resolve mitochondrial ETC complexes. Combined with histochemical staining, it has also been applied to measure ETC enzyme activities in muscles. The current study is to determine (1) whether BN-PAGE can be used to detect ETC complexes from different regions of the central nervous system (CNS) and (2) the quantitative range of BN-PAGE in measuring the amounts and activities of different ETC complexes. By systematically varying the protein amount and the time of histochemical reactions, we have found linear ranges comparable to spectrophotometric assays for measuring enzyme activities of several ETC complexes. In addition, we found linear ranges for measuring protein quantities in several ETC complexes. These results demonstrate that BN-PAGE can be used to measure the amount and activity of the ETC enzymes from the nerve tissues and, thus, can be applied to evaluate the functional changes of mitochondria in neurodegenerative disorders.
Jung
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Department of Biochemistry and Molecular Pharmacology, Department of Cell Biology, Neuroscience Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA.
Higgins
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CM
Xu
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Z
D000818:Animals; D002490:Central Nervous System / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D009410:Nerve Degeneration / Q000378:metabolism; D016548:Prosencephalon / Q000378:metabolism; D012394:Rosaniline Dyes; D013116:Spinal Cord / Q000378:metabolism
D012394:Rosaniline Dyes; C048139:Coomassie blue