How to separate the mitochondria
from the cell?
Experiment (1) to demonstrate
coupling
 Mitochondria are suspended in a buffered medium and an
O2 electrode monitors O2 consumption. At intervals,
samples are removed and assayed for the presence of ATP.
 Addition of ADP and Pi alone results in little or no
increase in either respiration (O2 consumption; black) or
ATP synthesis (red).
 When succinate is added, respiration begins immediately
and ATP is synthesized.
 Addition of cyanide (CN-), which blocks electron transfer
between cytochrome oxidase and O2, inhibits both
respiration and ATP synthesis.
Experiment (1) to demonstrate
coupling (Results)
Experiment (2) to demonstrate
coupling
 Mitochondria are suspended in a buffered medium
and an O2 electrode monitors O2 consumption. At
intervals, samples are removed and assayed for the
presence of ATP.
 Mitochondria provided with succinate respire and
synthesize ATP only when ADP and Pi are added.
 Subsequent addition of venturicidin or
oligomycin, inhibitors of ATP synthase, blocks both
ATP synthesis and respiration.
 Dinitrophenol (DNP) is an uncoupler, allowing
respiration to continue without ATP synthesis.
Experiment (2) to demonstrate
coupling (Results)
Experiment (3) Evidence for the role of
a proton gradient in ATP synthesis
 An artificially imposed electrochemical gradient can drive ATPsynthesis in the
absence of an oxidizable substrate as electron donor.
 In this two-step experiment,
 (a) isolated mitochondria are first incubated in a pH 9 buffer containing 0.1 M
KCl. Slow leakage of buffer and KCl into the mitochondria eventually brings the
matrix into equilibrium with the surrounding medium. No oxidizable
substrates are present.
 (b) Mitochondria are now separated from the pH 9 buffer and resuspended in
pH 7 buffer containing valinomycin but no KCl. The change in buffer creates a
difference of two pH units across the inner mitochondrial membrane. The
outward flow of K+, carried (by valinomycin)down its concentration gradient
without a counterion, creates a charge imbalance across the membrane (matrix
negative). The sum of the chemical potential provided by the pH difference
and the electrical potential provided by the separation of charges is a proton
motive force large enough to support ATP synthesis in the absence of an
oxidizable substrate.
Questions
1- DNP (2,4-dinitrophenol) and oligomycin are examples of inhibitors called
uncoupling agents. Uncouplers inhibit ATP synthesis but allow the other
reactions of cell respiration to proceed normally. Develop a hypothesis to
predict the effects of each agent on oxygen consumption.
2- Begin an experiment in Mitochondria Lab with mitochondria, pyruvate, and
ADP. Allow the reaction to proceed for one minute, then add an excess amount
of DNP by clicking on DNP and then clicking twice on the Add button. What
happens to oxygen consumption? Will the addition of more ADP influence
oxygen consumption? Add ADP and observe what happens. Explain your
answers. How might DNP be acting on the reactions of oxidative
phosphorylation to cause the change in oxygen consumption that you
observed?
3- What do you think is happening to the concentration of H+, ADP, and ATP in
this experiment? Repeat this experiment using oligomycin. What did you
observe?
Questions
4- 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.
5- 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?
Questions
6- 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?
Questions
7- 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