Experimental Rationale – Preparation for Polarographic Studies of Isolated Mitochondria

Research into the biology and biochemistry of cells and organelles relies heavily on biological models. We are interested in the utility of isolated liver mitochondria as a model system for addressing questions related to electron transport and oxidative phosphorylation. We can study mitochondrial function by measuring the rates at which they consume oxygen on different substrates and how exposure to reagents such as metabolic poisons affects oxygen consumption. A satisfactory model must give us predictable results. For isolated mitochondria to serve our purposes, we should be able to predict changes to oxygen consumption rates following an experimental intervention, based upon current theory.

Our study will require that we prepare isolated mitochondria from fresh liver tissue. We will then conduct polarographic studies on our preparations. Polarography is the term that we apply to measuring changes in dissolved oxygen. To prepare for this study you should first examine the pages on theory. If you have good recollection of college level introductory biology, then much of the information should be a review for you. You might nevertheless discover that you harbored a misconception or two. To prepare for the laboratory work you must look over the material on mitochondria in vitro. Following such preparation you should be ready to to think about the questions listed below.

A series of polarographic studies on isolated mitochondria is outlined on the following page. You should be prepared to predict what will happen following each addition of a substrate, ADP, or poison. To make such predictions you must work with known theory including specific paths taken by electrons in electron transport from a given substrate, proton translocation sites that are encountered along the way, when ATP synthase is or is not activated, and where inhibitors act or likely act. You must understand the underlying mechanisms. When you conduct the experiments you can then observe whether or not isolated mitochondria behave predictably. You will then be prepared to write up an analysis of the model, including its predictabillity and its shortcomings.

Mechanisms behind the effects of substrates, ADP, inhibitors, oligomycin, and uncoupling agents on oxygen consumption are not always obvious. Various rate-limiting factors and other complications, both biological and experimental, can influence the results. Consider how such inconsistencies must have frustrated Dr. Krebs and colleagues as they worked out the cyclic nature of mitochondrial oxidation of substrates and related mechanisms. In retrospect, the deductions seem to be a simple matter, but the investigators of the time could not simply purchase a Clark electrode or oxygen monitor from a laboratory supplier. They couldn't consult textbooks for 'maps' of the ETS or the sequence of Krebs reactions. Elucidation of the complex process by which mitochondria produce ATP required patience, dedication, insight, luck, sacrifice, and hard work – all of the above – by a great many investigators. I do hope you appreciate all of the trouble they have saved you.

Questions to consider

Here are some questions to consider as you prepare for the experiments and, later, as you write your discussion. A good discussion will address some of these questions as the results are interpreted, but they are not designed to be answered directly as if you were writing an essay. The object is to help guide your thinking, particularly in regard to the mechanisms behind the observed phenomena and the expected scope and depth of the analysis. Consider the significance of the mechanisms as well, that is, what purpose is served by the processes that you observe.

Mechanisms behind observed oxygen consumption

• Upon addition to the chamber isolated mitochondria will rapidly deplete the oxygen, but the depletion will be transient. Why?
• After initial depletion of oxygen, isolated mitochondria usually consume oxygen but at a slow pace. Why?
• What are the mechanisms behind state IVand state III respiration, respectively?
• What is the cause and effect relationship between the chemiosmotic gradient and electron transport (i.e., respiratory control)?
• Can ADP have any effect on uncoupled mitochondria? How about an electron transport inhibitor? Could changing substrates restore respiratory control?

Factors affecting measured oxygen consumption rates

• What mechanism should control the rate of oxygen consumption in state IV respiration? How about state III? Should the mechanism be different depending on the substrate? What if the gradient is destroyed, say, by adding an uncoupling agent?
• What is the predicted relationship (in terms of speed) among state IV, state III, and uncoupled electron transport rates on a given substrate? Why?
• Why might the state IV rate be different before and after adding ADP?
• What factors, either experimental or biological, might lead to discrepancies between predicted and actual ADP:O ratios?
• To interpret results on a given substrate do you have consider the fate of products of the initial reaction? Why or why not?
• Should you compare respiration rates as µmoles oxygen consumed per minute, or µmoles oxygen consumed per minute per µl mitochondria suspension? Why?
• Should rotenone completely stop state IV respiration with glutamate as substrate? Why might the inhibition be incomplete? Will addition of ADP stimulate a faster rate on glutamate in the presence of rotenone?
• What response(s) do you predict upon adding succinate to mitochondria that were exposed to rotenone then ADP?

Consequences of different electron transport pathways

• Should oxygen consumption rates be different for the three substrates that we use in these experiments?
• Can you completely discuss reasons for differences in rates or effects of inhibitors without tracing the path of electrons from a given substrate or identifying sites at which protons were translocated?
• Ideally, is an ADP:O ratio an integer?
• For well coupled mitochondria what ADP:O ratio would you predict for state III respiration on each substrate?
• What should the ADP:O ratio for ascorbate/TMPD tell you about where TMPD donates its electrons?
• Should the rate of respiration itself have any bearing on the ADP:O ratio?
• How can you manipulate the addition of substrates and inhibitors to determine where they bind?

Mechanisms of action of poisons

• What are possible causes of an uncoupling effect, what do they have in common, and what are the consequences of uncoupling?
• How is oligomycin known to inhibit oxidative phosphorylation? How can you test the predictive value of isolated mitochondria with regard to the action of oligomycin?
• Where does malonate bind? How is it known to exert its effect? Should it affect glutamate supported respiration?
• Once you add cyanide to the system, can you add any other reagent or combination of reagents to restore oxygen consumption?