In many experiments, it is preferable to measure mitochondrial function in whole cells, without the.

In many experiments, it is preferable to measure mitochondrial function in whole cells, without the.
In many experiments, it is preferable to measure mitochondrial function in whole cells, without the need to isolate the mitochondrion. Researchers use a sequence of compound injections to define the oxygen consumption of distinct mitochondrial states in intact respiring cells. Note that modern instruments directly plot oxygen consumption rates (OCR) rather than the oxygen concentration in the chamber. In a typical whole cell respirometry experiment (as shown in Figure A), The ATP synthase inhibitor Oligomycin is injected first to determine the level of oxygen consumption dependent on ATP phosphorylation. FCCP is injected after oligomycin to measure the maximal oxygen consumption level of the ETC independent of ATP phosphorylation. FCCP shuttles protons back into the matrix, thus bypassing ATP synthase entirely. The complex III inhibitor Antimycin is injected last to reveal the level of non-mitochondrial oxygen consumption from cytosolic enzymes such as NADPH oxidase. The sequential addition of compounds in a standard assay is shown in Figure A and the effect of each compound is illustrated in Figure B. The two questions that follow ask you to consider data from a whole cell respirometry experiment. Figure A. Oxygen consumption rate (OCR) vs time in intact cells. Figure B. Diagram of ETC and ATP synthase illustrating the effects of oligomycin, FCCP, and antimycin. DPHS is a compound that causes mitochondrial dysfunction. You perform a respirometry assay with cells treated or untreated with DPHS. To determine whether DPHS affects respiration by disrupting the metabolism of a specific fuel source or by disrupting electron transport, you perform parallel assays with glucose and fatty acids as different fuel sources. You obtain the following data. What do you conclude based on the respirometry data with glucose and fatty acids? Select ONE option: 1. DPHS disrupts electron transport because FCCP-stimulated maximal respiration is reduced with both glucose and fatty acid fuels. 2. DPHS increases proton leak because oligomycin-resistant respiration is increased with both fuels 3. DPHS specifically disrupts glucose metabolism because respiration of DPHS-treated cells is lower than untreated cells only when glucose is used as a fuel and there is no difference in respiration when fatty acids are used as a fuel. 4, DPHS specifically disrupts fatty acid metabolism because respiration of DPHS-treated cells is lower than untreated cells only when fatty acids are used as a fuel and there is no difference in respiration when glucose is used as a fuel.   Mar 31 2022 10:34 AM

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