Much of our knowledge of mitochondrial function results from the study of toxic compounds. Specific inhibitors were used to distinguish the electron transport system from the phosphorylation system and helped to define the sequence of redox carriers along the respiratory chain. If the chain is blocked then all the intermediates on the substrate side of the block become more reduced, while all those on the oxygen side become more oxidised. It is easy to see what has happened because the oxidised and reduced carriers often differ in their spectral properties. If a variety of different inhibitors are available then many of the respiratory carriers can be placed in the correct order.
There are six distinct types of poison which may affect mitochondrial function:
1) Respiratory chain inhibitors (e.g. cyanide, antimycin, rotenone & TTFA) block respiration in the presence of either ADP or uncouplers.
2) Phosphorylation inhibitors (e.g. oligomycin) abolish the burst of oxygen consumption after adding ADP, but have no effect on uncoupler-stimulated respiration.
3) Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP) abolish the obligatory linkage between the respiratory chain and the phosphorylation system which is observed with intact mitochondria.
4) Transport inhibitors (e.g. atractyloside, bongkrekic acid, NEM) either prevent the export of ATP, or the import of raw materials across the the mitochondrial inner membrane.
5) Ionophores (e.g. valinomycin, nigericin) make the inner membrane permeable to compounds which are ordinarily unable to cross.
6) Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate) which block one or more of the TCA cycle enzymes, or an ancillary reation.
Some of the best-known compounds are listed below:
Uncouplers & Ionophores: All of these compounds are small amphipathic molecules which dissolve in phospholipid bilayers and enormously increase their ionic permeability. They shield the electric charge as the ion passes through the membrane, providing a polar environment for the ion and a hydrophobic face to the outside world. Ionophores transport a variety of ions, but uncoupling agents specifically increase the proton permeability, and disconnect the electron transport chain from the formation of ATP. Some ionophores are natural products isolated from micro-organisms, but others are synthetic compounds, tailored to a specific application. The uncoupling agents are all synthetic, although there is a delicately regulated uncoupling protein involved in thermogenesis in brown adipose tissue mitochondria.
With intact mitochondria, CCCP enters in the protonated form, discharging the pH gradient, and then promptly leaves as the anion, destroying the membrane potential. The process can be repeated millions of times, so that a tiny amount of CCCP can catalyse the movement of huge numbers of protons, and short-circuit the respiratory chain.
Ionophores can be divided in to channel formers (such as gramicidin) which form a tiny pore through the membrane, and mobile carriers which diffuse backwards and forwards across the membrane. The ionophores used to study mitochondria normally belong to the mobile carrier group. They show considerable ionic specificity because the ion must be accommodated within a confined space inside the carrier: there are potassium ionophores, calcium ionophores and so forth.
Valinomycin: This mobile carrier catalyses the electrical movement of K+ across phospholipid bilayers. This implies that the potassium distribution across the membrane obeys the Nernst equation once equilibrium has been attained.
It is a cyclic amide/ester in which the sequence D-hydroxy- isovalerate, L-valine, L-lactate and D-valine is repeated three times (-A-B-C-D- on the diagram). The ionophore provides a polar interior to accommodate the potassium ion, but presents a non-polar lipophilic exterior to the outside world.
In some cases (e.g. valinomycin) the ionophore - ion complex has a net electrical charge, whereas the empty carrier is neutral. Other ionophores (e.g. nigericin) only form electrically neutral complexes. Charge makes an enormous difference to the behaviour of each carrier, since the charged complexes interact with any membrane potentials, whereas the neutral complexes are unaffected. We speak of electrical and electroneutral carrier mechanisms to draw attention to this difference.
This concept can be extended to cover the naturally occurring transport proteins in the inner mitochondrial membrane. Many of these proteins (and some ionophores) actually catalyse a swop of one ligand for another, hence the term antiporters. We also speak of symports (two molecules travel together in the same direction) and uniports (one molecule travels on its own.) Once again, the precise charge stoichiometry has a huge influence on the results.
Non-shivering thermogenesis: The mitochondria found in brown adipose tissue contain a unique uncoupling protein called thermogenin, which allows the controlled entry of protons without ATP sythesis in order to generate heat. The protein is a 33kd dimer, structurally related to the adenine nucleotide porter. It is inhibited by GTP, and activation is controlled by the sympathetic nervous system. This process is particularly important in new-born babies, which can lose heat very rapidly to their surroundings, but also occurs in adults and in hibernating animals. In contrast to the more familiar white adipose tissue, brown fat has an excellent capillary blood supply and can achieve very high metabolic rates. The major depot in humans is behind the shoulder blades, with other patches along the spine. The brown colour arises from the respiratory enzymes.
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