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Introduction/
training

• organization of the study
• polarography
• calibrating research paper

Mitochondria theory

• overview
• structure
• Krebs reactions
• electron transport
• the gradient
• oxidative phosphorylation

Mitochondria in vitro

• preparation
• fate of substrates
• state IV
• state III
• metabolic poisons
• mitotraces
• rationale
• experiments

Additional topics

• glossary of terms
• Hans Krebs
• origin of mitochondria
• other functions

Material culled from mitochondria pages

In an attempt to be concise and understandable, introductory level courses and textbooks frequently present concepts that are technically correct, but lead to misconceptions on the part of the student because they omit too much. In discussions about mitochondria students frequently come away with a superficial understanding of the true nature of Krebs' cycle, electron transport, respiratory control, and oxidative phosphorylation.

These pages were designed to supplement laboratory work with mitochondria by providing background in as much detail as the student might wish. Students at any level are likely to encounter terms with which they are unfamiliar. A glossary of terms is included in this project for your convenience. An overview of mitochondria structure and function is presented just to get you started. You can then wind your way through the main path of detailed information.

Although it is an oversimplification, you can picture the gradient as a buildup of protons in the intermembrane space. To the extent that protons leave the space they can be replaced by the ETS. A possible route for relief of the gradient is escape of protons from the outer membrane. As you may know, the outer membrane is porous to nearly anything with molecular weight less than 10,000 daltons (one dalton is the weight of a hydrogen atom). However, the membrane may act as a 'breakwater' that restricts the escape of protons despite its porosity. As long as there is substrate present, electron transport continually maintains the gradient, so the chemiosmotic gradient can be maintained despite a steady loss of protons through a 'leaky' membrane.

Mitochondria that have been poisoned by uncoupling agents or mechanically or chemically damaged cannot maintain a chemiosmotic gradient. That is, even with electron transport continuing at the maximum possible rate the protons that are translocated simply return or leave the intermembrane space by diffusion or convection, so that no energy gradient can be maintained at all. Undamaged, unpoisoned mitochondria in the presence of adequate substrate always maintain a fully 'charged' chemiosmotic gradient.

In a steady state, conditions are maintained constant although there may be considerable expenditure of energy in order to keep it so. The chemiosmotic gradient is maintained in a steady state, and so is the rate of electron transport in isolated mitochondria, once conditions have been established. It takes just a few moments for mitochondria to adjust to a change, such as the addition of substrate (probably due to the time it takes to take up the substance). The slopes you measure are steady state slopes, that is, the gradient is maintained and the rate of electron transport is constant.

For isolated mitochondria to conduct electron transport at a measurable rate, the substrate should support an efficient reaction that is coupled with the reduction of NAD or FAD. If one or more reactions or processes intervene before electrons can be donated to the ETS, the maximum possible rate will be slowed by the slowest step in the process. In a reaction sequence, this would be the step for which the substrate was least concentrated thus least available to the enzyme. If the rate at which energy can be delivered to the ETS is so slow that it cannot keep up with the loss of energy from the gradient, then the gradient cannot be maintained. Such is usually the case with fatty acids that remain in a liver mitochondria suspension. The concentration is too low to permit efficient delivery of acetyl-coenzyme A.

Is the presence of a rate-limiting step the reason for the difference in oxygen consumption rates between glutamate and succinate? To obtain an answer, consider the process that actually limits state IV respiration. Is it delivery of the substrate? Remember that to maintain a chemiosmotic gradient, energy must be supplied by the ETS 'on demand.' That is, when the energy level drops a tiny amount, the ETS replaces it, otherwise the gradient cannot be maintained. In the expanded definition of state IV respiration, the state IV rate is determined by the rate at which energy is lost from the gradient. There can be only one rate-limiting step in a sequence of steps.

If the state IV rate on glutamate is slow due to a rate-limiting step in the delivery of electrons to the ETS, then according to the reasoning outlined above there would be no chemiosmotic gradient. Adding ADP, then, would have no effect. Does it? The alternatives are that glutamate-supported respiration is slower in state IV than succinate-supported respiration either because the rate of loss of energy by the gradient is lower with glutamate, or else on glutamate the energy is replaced more efficiently, that is, less oxygen is used to pump the same number of protons across the inner membrane. Which is the more probable explanation?

Experimentally, you can demonstrate state III respiration by adding adenosine diphosphate (ADP) to a preparation in the presence of a suitable substrate. ADP binds the enzyme ATP synthetase, introduced in the overview. The binding of ADP 'unlocks' a channel that permits protons to flow into the matrix from outside the inner membrane. The entry of protons is coupled to the binding of ADP to inorganic phosphate, which also binds the enzyme complex. As the protons cross the inner membrane, energy in the gradient is removed, allowing the electron transport chain to speed up. The result is an increase in electron transport (and, consequently, oxygen consumption) and the synthesis of ATP. The rate of ADP-stimulated respiration in isolated mitochondria is called state III respiration. The synthesis of ATP by mitochondria is, of course, called oxidative phosphorylation.

In an isolated system, the mitochondria return to state IV respiration when the ADP is used up. The state IV rate may be higher than before, due to an uncoupling effect by ADP itself.

It must be emphasized that in living cells there is no such thing as state III or state IV respiration. Some of the energy in the gradient is lost as heat as protons escape via nonspecific mechanisms. Some energy is used for symport processes as described elsewhere. Some energy is used to make ATP. The degree to which each process takes place depends on cellular demand. Functions of mitochondria are subject to regulation just as are other cellular processes.

[uncoupling of mitos by ADP] One stumbling block to this explanation is that mitochondria in vitro might be ATP-depleted anyway. Nevertheless, adding an overwhelming amount of ADP astronomically shoots up the ratio of ADP to whatever ATP is indeed present, stopping any and all ATP-dependent reactions.

Here are the details for a system that employs Yellow Springs Instruments 5300 oxygen monitors, with #5331 standard oxygen probe.

The chamber should be filled with the medium to be used and allowed to equilibrate with stirring. The delivery of oxygen to the electrode must be steady, so the stir rate should be rapid, but not so rapid as to cause the stir bar to bounce. The latter condition interupts the diffusion of oxygen through the electrode membrane, resulting in a bumpy recording. A chamber is filled when the liquid completely fills all air spaces. In chambers with a glass stopper with capillary bore, the liquid should rise to the point at which a vortex is prevented when stirring. The chamber should be left un-stoppered when equilibrating, however, to allow air exchange.

YSI monitors are equipped with a digital display of % oxygen. The monitor should be warmed up for about 15 min before use. In O2 mode it should read 20.9% when the chamber is equilibrated with room air. In Air mode it should read100%. Since one should be able to convert to micromoles O2 consumed, it is convenient to use the Air mode, so that consumption of oxygen is recorded as a percentage of total oxygen in the chamber at the start of the experiment. If the chamber does not read 100% the locking ring on the CAL knob should be loosened (CCW), the CAL knob adjusted to set the readout to 100%, and the locking ring retightened.

To calibrate a chart recorder, the input should be grounded (most have a 'zero' button that does just that), and the pen position adjusted to zero. A data acquisition system should be equipped with 'zero' feature also. With the recorder set to a very low paper speed (0.1 mm/sec or less - to conserve paper), the output of the monitor should be recorded until zero slope is obtained. The YSI monitors we use deliver a 1 volt output at 100% saturation, so full scale on the recorder should be set to 1 volt (there should be a calibrated 1 volt setting). Some investigators prefer to set 100% at 95% of the full chart scale, so the pen doesn't 'peg' if oxygen content increases slightly.

The system is now calibrated. To record, an appropriate pen speed is selected (0.2 mm/sec with our Kipp & Zonen recorders), and measurements can begin. The pen doesn't have to be perfectly steady in order to start an experiment. In fact, it may wobble a bit when saturation is reached.

Oxygraph Performance Test