Assignment 4

1 .		Begin an experiment in MitochondriaLab with mitochondria, ADP, and glutamate. Allow the experiment to proceed for one minute, then add rotenone. Note: Keep a record of all your experiments in your lab notebook so you can refer back to your results as you interpret the results from other experiments. What happened to oxygen consumption? Now add ADP to the flask. What happened to oxygen consumption after adding ADP? Is this what you expected? Explain your answers in the field below.
2 .		Ascorbate can bind to a complex in the electron transport chain and donate electrons to the chain. Use ascorbate to help you pinpoint where rotenone is blocking the chain by repeating the same experiment with mitochondria, ADP, and glutamate; then wait one minute, add rotenone, wait another minute, and then add ADP and ascorbate/TMPD to the experiment. What happened to oxygen consumption? Can you determine which complex of the electron transport chain is bound by ascorbate? Based on these results and knowledge of where ascorbate binds, can you determine which complex (I, II, III, or IV) is likely to be inhibited by rotenone?
3 .		Succinate can also bind to a complex in the electron transport chain and donate electrons to the chain. Use succinate to help you determine where rotenone is blocking the chain by repeating the same experiment as above with mitochondria, ADP, and glutamate; then wait one minute, add rotenone, wait another minute, and then add ADP and succinate to the experiment. What happened to oxygen consumption? Can you determine which complex of the electron transport chain is bound by succinate? Based on these results and knowledge of where succinate binds, can you determine which complex (I, II, III, or IV) is likely to be inhibited by rotenone. Based on your results and knowledge of where succinate binds, which complex (I, II, III, or IV) is likely to be inhibited by rotenone?
4 .		Antimycin is another metabolic inhibitor that acts on the electron transport chain. Use antimycin to help you pinpoint where rotenone is blocking the chain by repeating the experiment in step (b) but after adding rotenone, add ADP and antimycin to the experiment. What happened to oxygen consumption? Which complex of the electron transport chain is bound by antimycin? Based on your results and knowledge of where antimycin binds, which complex (I, II, III, or IV) is likely to be inhibited by rotenone? Are all of your results consistent enough to help you pinpoint the binding site of rotenone? Once you have determined which complex is bound by rotenone, discuss your answer with your instructor to find out the specific molecules affected by rotenone, ascorbate, succinate, and antimycin.