June 25,2010 – submit one journal article at the admi office (request them to shove it in my pigeon hole) related to biochem which you wish to share on the time of your journal flash report (year 2000 to present only)

July 1, 2010 topic on nutrition

Validation on July 8, 2010

See assigned group topics after the reading for major report.

BRIEF BACKGROUND

Cytochrome spectra from the respiratory pigments in animal tissues were originally observed in the nineteenth century, but dismissed as an artefact by the scientific establishment of the day. Oxygen uptake by tissue homogenates, and the action of simple respiratory inhibitors were first studied systematically in the 1930s, when it was realised that electrons flowed from substrates to oxygen via a sequence of redox carriers, several of which showed distinctive spectral changes when oxidised and reduced.

The isolation of intact mitochondria followed improvements in centrifuge technology in the 1940s, and the introduction of isotonic sucrose preparation media to prevent osmotic lysis. Investigators noted the phenomenon of latency: the internal mitochondrial enzymes could not be demonstrated until the organelles had been broken open. The basic structure of mitochondria was established by electron microscopy and it was shown by controlled disruption that most of the respiratory enzymes responsible for oxygen uptake were attached to the inner mitochondrial membrane (which is extensively folded to form the mitochondrial cristae) while the soluble enzymes catalysing the Krebs cycle and fatty acid oxidation pathways were confined to the internal matrix space.

This electron microscope picture of a chick embryo mitochondrion is used by kind permission of Professor Ruth Bellairs, Department of Anatomy and Developmental Biology, University College, Gower Street, London WC1E 6BT.

It was later shown that the inner membrane was a major permeability barrier within the cell, but the outer membrane contained a protein called porin which rendered it largely largely permeable to molecules less than about 1500 daltons. An important group of enzymes which metabolise ATP, including myokinase, creatine kinase, and the nucleoside diphosphate kinases, are trapped in the inter-membrane space, between the inner and the outer membranes.

myokinase: ATP + AMP <=> ADP + ADP

creatine kinase: ATP + creatine <=> ADP + creatine phosphate

cytidine diphosphate kinase: CDP + ATP <=> CTP + ADP (many similar enzymes exist)

Experimental work accelerated with the introduction of the more convenient Clarke-type oxygen electrode in the 1950's in place of the older manometric methods. It was shown that the uptake of oxygen by intact mitochondria depended on the simultaneous conversion of ADP and inorganic phosphate into ATP. The phenomenon is termed respiratory control. The obligatory dependence of a highly favourable chemical reaction (substrate oxidation) on the simultaneous execution of an unrelated and extremely unfavourable reaction (ATP synthesis) was immediately recognised as a novel and important concept by the biochemists of the day.

Oxygen uptake which is dependent on the presence of ADP and phosphate is termed coupled respiration. The coupling of respiration and phosphorylation could be broken by mechanical or

osmotic disruption of the mitochondria, or by the addition of uncoupling agents, in which case respiration proceeded rapidly without any concomitant ATP synthesis. The original uncoupling agent was 2,4-dinitrophenol, which has now been replaced by more effective compounds such as FCCP (para-trifluoromethoxy carbonyl cyanide phenylhydrazone) and CCCP (meta-chloro carbonyl cyanide phenylhydrazone).

Two major classes of mitochondrial inhibitor could be distinguished: respiration inhibitors and phosphorylation inhibitors. Both types were effective against intact mitochondria, but only respiration inhibitors could prevent oxygen uptake after the addition of an uncoupling agent. These experiments gave rise to the concept of an energetically favourable electron transport system, which in some way powered an energetically unfavourable phosphorylation system. The free energy available from the redox reactions was used to drive ATP synthesis. Respiration inhibitors such as cyanide ions blocked the electron transport chain and were always effective in suppressing oxygen uptake, whereas phosphorylation inhibitors such as oligomycin, which prevented ATP manufacture, could only block respiration if the coupling were intact.

From this point, in the mid 1950s, the subject diverted into a long and occasionally acrimonious blind alley, as scientists attempted to find a chemical intermediate linking respiration to ATP generation. This idea derived from the substrate level phosphorylations observed in the glycolytic pathway. This search was unsuccessful as no such intermediate exists. We now know that the energy from respiration is first captured in the form of pH and electrical potential gradients across the inner mitochondrial membrane. The energy stored in these gradients is later exploited to drive the synthesis of ATP. This chemiosmotic theory was first proposed in 1961 by a British biochemist, Peter Mitchell, who worked for many years without much official recogonition or significant public funding until he was awarded the Nobel Prize for Chemistry in 1978.

OXYGEN ELECTRODES

Much of our knowledge of electron transport in mitochondria and chloroplasts comes from oxygen electrode recordings. The oxygen concentration in a sealed incubation chamber is continuously monitored, and the effects of making various additions to the chamber can be observed. A cross section through a typical apparatus is shown below:

The upper section containing a transparent, thermostatted sample chamber is secured to the lower electrode assembly with a screwed ring. A thin teflon membrane is trapped between the two sections and separates the isotonic incubation medium from the strong KCl electrolyte in the electrode compartment. The adjustable stopper is used to seal the incubation chamber and prevent room air dissolving during the experiments. The small hole in the centre of the stopper permits the expulsion of air bubbles and allows small

volumes of reagents to be added with a microlitre syringe. The contents of the chamber are stirred continuously with a magnetic flea.

A small polarising voltage (ca. 0.6 volt) is applied between the silver anode (+) and the platinum cathode (-). Oxygen diffuses through the teflon membrane and is reduced to water at the platinum cathode:

O2 + 4H+ + 4e-1 = 2H2O

The circuit is completed at the silver anode, which is slowly corroded by the KCl electrolyte:

Ag + Cl- = Ag Cl + e-1

The resulting current is proportional to the oxygen concentration in the sample chamber. This signal can be amplified and recorded.

The whole process is totally dependent on the supply of oxygen. The rate of oxygen diffusion to the cathode (and hence the current) depends on the oxygen concentration in the main incubation chamber. It also depends on several other factors: temperature, membrane thickness and permeability, sample viscosity and stirring speed. In contrast to pH electrodes which measure an equilibrium position, oxygen electrodes measure the velocity of a physico-chemical process that is far from equilibrium. pH electrodes have an intrinsic thermodynamic response which is relatively insensitive to temperature and sample composition, but there is no intrinsic calibration to an oxygen electrode - at regular intervals, or if the instrument is dis-assembled, it must be re-calibrated against a known standard, usually air. It is particularly important to control the temperature.

OXYGRAPH RECORDINGS

If mitochondria are incubated in an oxygraph apparatus (oxygen electrode) in an isotonic medium containing substrate and phosphate, then addition of ADP causes a sudden burst of oxygen uptake as the ADP is converted into ATP:

The actively respiring state is sometimes refered to as "state 3" respiration, while the slower rate after all the ADP has been phosphorylated to form ATP is refered to as "state 4".

State 4 respiration is usually faster than the original rate before the first addition of ADP

because some ATP is broken down by ATPase activities contaminating the preparation, and the resulting ADP is then re-phosphorylated by the intact mitochondria.

The ratio [state 3 rate] : [state 4 rate] is called the respiratory control index and indicates the tightness of the coupling between respiration and phosphorylation. With isolated mitochondria the coupling is not perfect, probably as a result of mechanical damage during the isolation procedure. Typical RCI values range from 3 to 10, varying with the substrate and the quality of the preparation. Coupling is thought to be better in vivo, but may still not achieve 100%.

It is possible to calculate a P:O ratio (the relationship between ATP synthesis and oxygen consumption) by measuring the decrease in oxygen concentration during the rapid burst of state 3 respiration after adding a known amount of ADP. It is necessary to subtract the basal respiration due to imperfect coupling and the recycling of ATP, as shown in the graph above. The change in concentration must then be multiplied by the chamber volume, so that the answer (in micro-atoms of oxygen) can be related to the quantity of ADP added. The quantity of oxygen in the chamber is calculated from published oxygen solubility data at the appropriate temperature. Using the figures from the graph above:

decrease in O2 (from graph) = 0.135 mM; chamber volume = 2.5ml

oxygen atoms consumed in state 3 = 0.135 * 2 * 2.5 = 0.68 micro-atoms (note the factor of 2, allowing for 2 atoms in an oxygen molecule)

injected ADP (20 microlitres of a 50mM solution) = ATP formed = 1 micromole in total

P:O = [ATP formed] : [oxygen consumed] = 1.48 (for succinate oxidation)

NAD-linked substrates give consistently higher values for the P:O ratio (about 2.5) compared with succinate (about 1.5). These results indicate that electrons from relatively poor reducing agents such as succinate (also acyl CoA and glycerol phosphate) enter part of the way along the respiratory chain, by-passing the first coupling site where energy is captured for ATP synthesis.

The sharp changes in slope after exhaustion of ADP and again after all the oxygen has been used up imply that mitochondria must have very high affinities for ADP and oxygen. The concentration of both these compounds is very low in most healthy cells, since they are efficiently scavenged by the mitochondria.

The addition of an uncoupling agent (such as dinitrophenol or CCCP) leads to a permanently high rate of respiration in the absence of ADP, until all the oxygen has been consumed.

Many natural and synthetic poisons block mitochondrial respiration. If mitochondria are incubated in an oxygraph experiment with substrates and inorganic phosphate, the interactions between inhibitors and uncouplers allow two major types of inhibition to be distinguished:

Mitochondrial respiration ratesadditions / inhibitors none oligomycin cyanide

none low low zeroADP high low zerouncoupling agent high high zero

The conclusion to be drawn from this type of experiment is that cyanide prevents respiration by blocking the respiratory chain itself, so cyanide is effective whether or not ADP or uncoupler are added. Oligomycin on the other hand inhibits the ADP phosphorylation system, so it only blocks respiration in coupled mitochondria.

RESPIRATORY CHAIN COMPONENTS

The system of mitochondrial enzymes and redox carrier molecules which ferry reducing equivalents from substrates to oxygen are collectively known as the electron transport system, or the respiratory chain. This system captures the free energy available from substrate oxidation so that it may later be applied to the synthesis of ATP. Many respiratory chain components were first identified in crude homogenates through their spectral properties, which frequently change when a carrier is oxidised or reduced. Fractionation of mitochondria in the presence of mild detergents or chaotropic salts dissected the respiratory chain into four large multi-subunit complexes containing the principal respiratory carriers, named Complex 1 to Complex 4. These substantial protein "icebergs" float in the sheet of inner membrane lipids, often presenting one face to the mitochondrial matrix and another to the inter - membrane space. Many of their components have now been isolated in a relatively pure form. Other membrane bound enzymes such as the energy linked transhydrogenase (ELTH) are also present which fulfil ancillary roles.

The main components participate in the approximate order of their redox potentials, and the bulky complexes are linked by low molecular weight mobile carriers which ferry the reducing equivalents from one complex to the next. Except for succinate dehydrogenase (complex 2) all these complexes pump protons from the matrix space into the cytosol as they transfer reducing equivalents (either hydrogen atoms or electrons) from one carrier to the next. The diagram above shows the flow of reducing equivalents in purple, and movement of the positively charged protons in red. Proton pumping is an arduous task which creates substantial pH and electrical gradients across the mitochondrial inner membrane. These protons eventually re-enter the matrix space via the F1 ATPase, driving the synthesis of ATP as they return.

The number of protons and the number of positive charges crossing the inner membrane need not necessarily agree for each individual transmembrane protein, although the accounts must balance for the whole ensemble. This discrepancy is illustrated on the diagram above, and is explained in greater detail below.

The sequence of of the carriers in the respiratory chain was clarified by the use of specific inhibitors to block the flow of electrons from substrates to oxygen. All components on the substrate side of a block become more reduced, while those which follow become more oxidised. These changes can often be observed spectroscopically. If a range of inhibitors are available, acting at different places, then their precise points of action and the order of the carriers is defined.

Further information was gleaned from the use of artificial redox mediators. Electron donors, such as ascorbate [vitamin C] plus the synthetic compound tetramethyl phenylene diamine can contribute electrons to cytochrome c, while artificial acceptors such as ferricyanide would collect electrons from complex 1 or complex 2 and by-pass the remainder of the chain. This could be particularly informative when used in conjunction with specific inhibitors, or when ATP yields were measured for particular segments of the chain.

Respiratory Chain Components[click for additional details]

Location Prosthetic     groups

Function 

NADPH / NADP (almost 100% in the reduced form)

matrix space (separate cytosolic pool)

- mobile carrier

energy-linked transhydrogenase NADPH + NAD+

=> NADH + NADP+

membrane spanning protein

none proton pump 2H+/2e-1

NADH / NAD (less than 30% in the reduced form)

matrix space (separate cytosolic pool)

- mobile carrier

NADH dehydrogenase (complex 1)

membrane spanning multi-subunit protein

non-heme iron& FMN

proton pump 4H+/2e-1

succinate dehydrogenase (complex 2)   [see note 1]

membrane spanning multi-subunit protein

non-heme iron& FAD

no proton pumping

ubiquinol / ubiquinone dissolved in the inner - mobile carrier

membrane lipids

ubiquinol:cytochrome c reductase (complex 3)

membrane spanning multi-subunit protein

non-heme iron, heme b & heme c1

proton pump 4H+/2e-1

[see note 2]

cytochrome c (ferrous / ferric)

inter-membrane space

heme c mobile carrier

cytochrome c oxidase (complex 4)

membrane spanning multi-subunit protein

copper, heme a& heme a3

proton pump 2H+/2e-1

[see note 2]

F0 / F1 ATPase(ATP synthetase)

membrane spanning multi-subunit protein

none proton pump3H+ / ATP

Note 1: In addition to succinate dehydrogenase, several other enzymes contribute electrons to directly to ubiquinone. Acyl CoA dehydrogenase is a soluble flavoprotein involved in fatty acid oxidation in the mitochondrial matrix space. It feeds its reducing equivalents to ubiquinone via an intermediate low molecular weight electron transferring flavoprotein (ETF). Glycerol phosphate "oxidase" is another flavoprotein which also feeds reducing equivalents to ubiquinone. It is an inner membrane protein, but the relevant active centre faces outwards and reacts with its substrate in the cytosolic compartment, not in the matrix space. None of these enzymes contributes to proton pumping, so the P:O ratio from their substrates is only1.5

Note 2: The proton pumping stoichiometry for complex 3 and complex 4 is difficult because the electron acceptor for complex 3 (cytochrome c) is located on the outside of the inner membrane, whereas the oxidant for complex 4 (molecular oxygen) is reduced on the inside face of the inner membrane. It is necessary to count the negative charges on the electrons traversing the chain as well as the positive charges on the protons. Moreover, reduction of oxygen consumes two additional "chemical" protons to form water, in addition to those pumped by complex 4. These "chemical" protons don't count because they exactly balance the two "chemical" protons produced earlier when the hydrogen atoms were first removed during substrate oxidation. The net effect of all this is that complex 3 exports four protons but only two positive charges for each pair of electrons traversing the respiratory chain, while complex 4 exports two protons but four positive charges.

Liposomes are membranous vesicles which form spontaneously when many phospholipids are dispersed in aqueous media. If the lipids are simply shaken with water large multi-layered structures are produced with an "onion skin" arrangement. If the multi-layered liposomes are treated with ultrasonic vibrations they yield much smaller unilamellar vesicles with a simple limiting membrane composed of a single phospholipid bilayer. Both single and multi-layered liposomes can be prepared which are impermeable to the majority of charged ions, although

uncharged, hydrophobic molecules have ready access to the interior. A small volume of the original preparation medium becomes trapped within each liposome as the membrane seals up.

Reconstitution: If hydrophobic membrane proteins are included during the lipid sonication step, these are incorporated into the liposome membranes. This has been used to re-constitute the respiratory chain from purified fragments. Unfortunately the proteins adopt a random orientation as they are incorporated into the liposome membrane, so half of them finish facing the wrong way round. It is therefore difficult to re-create a proton gradient. A variety of ingenious techniques have been used to select the correctly oriented protein population, for example by trapping the electron donor within the liposome and supplying the acceptor only to the external face.

Liposomes are also being actively researched as a means of delivering cytotoxic drugs to tumour cells, which have been targeted with monoclonal antibodies.

UNCOUPLERS AND INHIBITORS

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:

Compound Mode of action and effects

Amino-oxyacetate

Ancillary enzyme inhibitor. Inhibits all transaminases by reacting covalently with their pyridoxal phosphate prosthetic group. Blocks the malate / aspartate cycle by inhibiting glutamate - oxaloacetate transaminase (GOT).

Antimycin A Respiration inhibitor. Blocks the respiratory chain at complex 3 between cytochrome b and cytochrome c1. It therefore prevents the oxidation of both NADH and succinate, but has no effect on ascorbate + TMPD.

Arsenite Krebs cycle inhibitor. Reacts with the disulfide linkage in oxidised lipoic acid, forming a cyclic adduct. Inhibits all the oxo-acid dehydrogenases, including pyruvate dehydrogenase, oxoglutarate dehydrogenase and the branched chain oxo-acid dehydrogenase.

Atractyloside Transport inhibitor. Blocks the adenine nucleotide porter by binding to the outward - facing conformation (contrast with bongkrekic acid). It has no effect on sub-mitochondrial particles, which re-seal spontaneously after sonication with the membranes inside-out. This ATP/ADP transport inhibitor resembles oligomycin when used with intact mitochondria. (See also Bongkrekic acid.)

Bongkrekic acid Transport inhibitor. Blocks the adenine nucleotide porter by binding to the inward- facing conformation (contrast with atractyloside).

Cyanide Respiration inhibitor. Blocks cytochrome oxidase (complex 4) and prevents both coupled and uncoupled respiration with all substrates, including NADH, succinate and ascorbate + TMPD.

Mersalyl Transport inhibitor. Dose dependent inhibition of the phosphate and dicarboxylate porters. Mersalyl is an outdated mecurial diuretic.

N-ethyl maleimide (NEM)

Transport inhibitor. Blocks the phosphate porter by reacting with -SH groups, and prevents respiration by coupled mitochondria and phosphate - mediated swelling.

Oligomycin Phosphorylation inhibitor. Binds to a 23kd polypeptide (OSCP) in the F0

baseplate and blocks ATP synthesis (and degradation) by the F0 /F1 ATPase. It abolishes ADP-stimulated respiration in intact mitochondria and all ATP-driven functions in sub-mitochondrial particles.

Rotenone Respiration inhibitor. Blocks NADH dehydrogenase (complex 1) in the respiratory chain but has no effect on the oxidation of either succinate or ascorbate + TMPD. (TMPD is tetra methyl phenylene diamine, an artificial redox mediator which assists the transfer of electrons from ascorbate to cytochrome c.)

Thenoyl trifluoro acetone (TTFA)

Blocks succinate dehydrogenase (complex 2). It has no effect on the oxidation of NADH-linked substrates or ascorbate + TMPD.

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.

CCCP: (carbonyl cyanide m-chloro phenyl hydrazone) This is a lipid-soluble weak acid which is a very powerful mitochondrial uncoupling agent. The compound FCCP (p-trifluoromethoxy carbonyl cyanide phenyl hydrazone) is similar.

The negative charge is extensively delocalised over about ten atoms in the ionised form of CCCP, so the electric field surrounding the CCCP anion is very weak. This allows the anion to diffuse freely through non-polar media, such as phospholipid membranes. This behaviour is very unusual: the vast majority of electrically charged ions are excluded from non-polar environments.

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.

Nigericin: This mobile carrier resembles valinomycin but contains a carboxyl group and forms a potassium salt. It therefore catalyses an electroneutral potassium/proton exchange across lipid bilayers. This implies that the potassium distribution will be related to the pH gradient once equilibrium has been attained:

[K+ ]in / [H+ ]in = [K+ ]out / [H+ ]out

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.

SUBMITOCHONDRIAL PARTICLES

Treatment of mitochondria with ultrasonic vibrations under appropriate conditions tears open the membranes and leads to the formation of tiny inside-out vesicles derived from the inner membrane, containing trapped cytochrome c originating from the inter-membrane space. Sub-mitochondrial particles may retain partial coupling between electron transport and ADP phosphorylation. They have been much used for experimental work because their inverted membrane orientation provides direct access to the respiratory chain without the complications introduced by the substrate transport systems.

Two activities which are most easily studied in sub-mitochondrial particles (although they also occur with intact mitochondria) are the energy linked transhydrogenase and reversed electron transport.

The energy-linked transhydrogenase: This bizarre membrane-spanning enzyme catalyses the reversible transfer of reducing equivalents between the mitochondrial NADPH + NADP pool and the NADH + NAD pool, while simultaneously pumping 2 protons and 2 charges across the inner mitochondrial membrane. The energy linkage keeps the NADPH/NADP couple about 500 times more reduced than the NADH/NAD couple, despite the exact equivalence of their standard redox potentials.

NADPH + NAD+ + 2H+(inside) <=> NADP+ + NADH + 2H+(outside)

It is not clear which direction the transhydrogenase takes in vivo. Enzymes reacting with NADP generally have a more negative redox potential than those reacting with NAD, except for glutamate dehydrogenase, which is the only enzyme with a dual coenzyme specificity. This is a problem, since the dual coenzyme specificity should lead to an energy-wasting futile cycle if the two coenzyme pools are at different redox potentials.

The arrangement must confer some selective advantage (having persisted unchanged for 2000 million years!) but the biological benefits remain obscure. It may involve the overall regulation of nitrogen balance, by using the ammonia-fixing reaction with NADPH to partially counteract the ammonia formation with NAD, but this hypothesis remains highly speculative. Mitchell once suggested that the NADPH-linked pathways for isocitrate, malate and glutamate were equivalent to fifth gear on a car, giving enhanced ATP yields and better fuel economy at the expense of

snappy performance. When maximum ATP flux was required the ADP-mediated activation of the NAD-linked pathways would "drop down a gear" to enter the overtaking lane.

Reversed electron transport: Oxidative phosphorylation is a partially reversible process and in the presence of an artificially high ATP/ADP ratio electrons from a weak reducing agent like succinate can be forced backwards through the respiratory chain carriers to yield a stronger reductant such as NADH:

succinate + NAD+ + energy => fumarate + NADH + H+     (overall reaction)

Ethanol is relatively poor reducing agent, and will only reduce a tiny proportion of any added NAD. However, in an artificial system containing sub-mitochondrial particles, added alcohol dehydrogenase, a trace of NAD and excess NADP, electrons from ethanol can be forced backwards via a small pool of NADH through the energy-linked transhydrogenase to form large amounts of the excellent reducing agent, NADPH. The reaction requires a source of energy: either added ATP, or respiration using another segment of the respiratory chain.

ethanol + NAD+ => acetaldehyde + NADH + H+

NADH + NADP+ + energy => NAD+ + NADPH

The process has little physiological relevance, but gave enormous insight into mitochondrial function. Reversed electron transport from ethanol to NADP in rotenone-blocked sub-mitochondrial particles can be driven either by energy from external ATP (reversing the normal operation of the F1ATPase) or by the energy from succinate oxidation via complex 2, complex 3 and complex 4. Both routes are sensitive to uncouplers, but oligomycin only blocks the process when it is driven by ATP. This key observation showed that there must be a common high energy intermediate between the coupling sites on the respiratory chain and the manufacture of ATP. The intermediate is known as the high energy pool.

THE HIGH ENERGY POOL

Reversed electron transport experiments lead to a realisation in the 1950s that there must be a common, non-phosphorylated high-energy intermediate between the respiratory chain carriers and the synthesis of ATP. The concept is illustrated below. The arrows show the normal direction of flow, but most of the reactions are freely reversible, and energy added to the system via one process can be utilised elsewhere. The high energy pool was originally thought to be a chemical compound, but nowadays we know that it is actually the pH and electrical gradients developed across the inner mitochondrial membrane.

It is possible to measure the gradients by studying the distribution of permeant molecules across the mitochondrial inner membrane. This requires a method for the rapid separation of mitochondria from their incubation medium, either by rapid ultrafiltration, or by centrifugation through a layer of silicone oil. An impermeable radioactive marker (commonly sucrose) is added to the incubation in order to correct for external medium contaminating the organelles.

Lipid soluble weak acids can be used to measure the pH differential. This method exploits the easy membrane permeability of the electrically neutral free acid, and the impermeability of its ionised salts. It is vital that the free acid crosses the membrane in the protonated form without net movement of electrical charge. At equilibrium the concentration of the free acid is the same everywhere, but the ratio of [free acid] to [ionised salt] differs in the two compartments. In the matrix space the amount of salt is proportional to the internal pH, but outside the mitochondria the ionised salt concentration depends on the external pH. If the internal and external salt concentrations can be measured then application of the Henderson Hasselbach equation yields the internal pH.

Measurement of the membrane potential ( ) requires a permeant cation, e.g. a radioactive alkali metal plus valinomycin, or the lipid soluble tetraphenyl phosphonium. In this case it is important that the cation penetrates the membrane with net movement of charge, so that the cation distribution is influenced by the membrane potential. If the internal and external concentrations of a permeant cation can be measured, then the membrane potential can be calculated from the Nernst equation.

Some dyestuffs change their absorbtion or fluorescence properties when they are aligned in a strong electric field. This provides an alternative method for the continuous measurement of mitochondrial membrane potential by adding such compounds to a mitochondrial suspension. Suitable compounds include carbocyanines, phenosafranine and bisoxonols. Although it is very convenient, this technique must be calibrated using the older, single point methods.

The sum of the membrane potential and the pH gradient are together known as the proton motive force (PMF). This indicates the total potential energy stored in the transmembrane gradients, which is available to drive protons back into the matrix space, and provide the power for biologically useful processes. Using the correct sign for the pH gradient (negative!) and substituting numerical values for the gas constant, faraday constant and the absolute temperature, it follows that:

PMF (in millivolts) = - 60 pH

at 37oC. The minus sign is confusing: the pH gradient is itself negative, so the two components are normally additive in their effects.

In mitochondria the electrical gradient, (150mV, inside negative) makes a larger contribution than the pH gradient (0.5pH units, inside alkaline). In chloroplasts pH is more important. The mitochondrial pH corresponds to a three-fold difference in hydrogen ion concentration between matrix and cytosol. For each transmembrane process, the pH and components may act either separately or together, depending on the enzyme structure and the balance of biological advantage. A proton movement would normally carry one positive charge, but other ions may move as well, so there is no need for the proton and charge counts to balance for any individual membrane transport process, although the totals must obviously balance overall.

The best current estimates for the proton and charge counts for the repiratory chain are shown in the section on redox carriers. For each pair of electrons to traverse the chain, NADH dehydrogenase moves 4 protons and 4 positive charges outwards across the inner membrane from matrix to cytosol, ubiquinol cytochrome c reductase moves 4 protons and 2 charges, while cytochrome oxidase moves 2 protons and 4 charges. The re-oxidation of NADH to form NAD and water is thus associated with the net export of 10 protons and 10 positve charges from the mitochondrial matrix to the cytosolic compartment.

ATP synthesis by the F1ATPase is thought to require 3 protons carrying 3 positive charges to re-enter the matrix space. In addition, 1 proton (but no charge) is needed for phosphate uptake and 1 charge (but no proton) for the ATP/ADP exchange with the cytosol (see the metabolite transport pages for details). Thus the overall requirement is 4 protons and 4 charges for each ATP delivered to the outside world. This implies that the P:O ratio is 2.5 for NAD-linked substrates, and 1.5 for succinate, which is rather lower than some older text-book values, but much closer to the actual experimental results!

The lipid bilayer in the inner membrane is only about 5nm thick and is therefore subject to enormous electrical forces:

150 x 10-3 volts across 5 x 10-9 metres = 30,000,000 volts / metre

The inner membrane has an unusual phospholipid composition which includes cardiolipin (diphosphatidylglycerol) and is particularly "leak proof" to small ions.

Most types of phospholipid will spontaneously form liposomes when agitated with water, and this has been exploited to re-constitute the respiratory chain from purified components.

The F0F1-ATPase

This substantial enzyme (Mr approximately 500,000) is readily visible in electron micrographs as 8.5 nm spheres attached to the matrix side of the mitochondrial inner membrane. The spheres can be detached by a variety of methods, after which they act as an ATPase. The physiological function of this enzyme is the synthesis of ATP, using the energy stored in the transmembrane pH and potential gradients.

The complete assembly contains at least 12 different types of polypeptide chain, several of which are present in multiple copies. The catalytic head group is connected by an oligomycin sensitive stalk to a proton conducting baseplate in the mitochondrial inner membrane. Three protons are thought to pass through the membrane from the external P phase to the internal N phase for each molecule of ATP manufactured by the complex.

The F1 head group contains three nucleotide binding sites, and the enzyme probably performs a three-phase catalytic cycle. In the first phase, ADP and phosphate bind to one active centre, which catalyses the formation of bound ATP. This step is energetically possible because the free energy released by tightly binding the ATP to the active centre compensates for the instability of the new phospho anhydride bond. The energy from the proton motive force is required to prise the ATP from the active centre.

The F0 base piece embedded in the mitochondrial inner membrane is a molecular turbine driven by the trans-membrane proton gradient. Proton entry forces a central camshaft to rotate within the F0 baseplate and the F1 head group, altering the subunit conformation as this movement takes place. A second, off-centre protein tether connects the head group to the base piece and prevents the head piece spinning uselessly as the central shaft rotates. Energy is transmitted to the catalytic subunits in the ATP synthase F1 headpiece by the rotation of the camshaft. The "cam" distorts the protein subunits, destroying their ability to bind ATP. The energy input is used to drive ATP release, not for bond formation.

It is presumably necessary to disable the catalytic mechanism on the centre which has just formed ATP (to stop this centre hydrolysing its own product) before destroying its ability to bind ATP. This allows the product to be released. Meanwhile, the two other active centres are performing their own parts of the catalytic cycle. The three active centres operate simultaneously, but 120o out of phase. It takes at least 9 protons (possibly as many as 12) to drive one revolution of the camshaft and produce 3 ATP molecules.

Remember that the whole complex is reversible. Normally the energy from the proton gradient is used to manufacture ATP, but it is equally possible in vitro to do things the other way round, and use the hydrolysis of ATP to drive the camshaft, and ultimately pump protons back through the turbine and into the extramitochondrial compartment. If the F0 base piece is not attached to a membrane nothing useful will be accomplished, and the complex will simply act as an ATPase, as was originally observed.

It is possible to directly observe the rotation of the F1ATPase cam shaft using a fluorescence microscope, although considerable ingenuity is required. Noji et al (1997) Nature 386, 299-302 used a genetically modified F1ATPase from a thermophilic bacterium expressed in E. coli. They discarded the F0 basepiece and tethered the F1 motor head groups to a glass plate using polyhistidine tags attached to the N-termini of all three beta subunits. The glass plate had been pre-treated with horseradish peroxidase conjugated with the nickel complex of nitrilotriacetic acid, to which polyhistidine binds with high affinity. [Nitrilotriacetic acid looks like half an EDTA molecule, so it leaves un-coordinated nickel positions available for external ligands.]

The motors were glued down by their large catalytic subunits, leaving the motor shafts exposed, and facing away from the glass. The gamma subunits which form the shaft were modified by site directed mutagenesis to remove the original Cys193 (which is inconveniently far down the shaft) and replace it with serine. These workers also replaced Ser107 in the stalk region with cysteine.

This single cysteine residue (the only one in the molecule) could then be biotinylated, and linked using streptavidin to fluorescently labelled, biotinylated actin filaments. [Streptavidin has four biotin binding sites.]

The fluorescent actin filaments were many times larger than the tethered motors and could be visualised in a light microscope.

Addition of 2mM ATP caused a small number of  the motor shafts marked by the actin filaments to rotate in a counter- clockwise direction. The movie shows the results they obtained.

Circular motion also occurs in the proteins which rotate bacterial flagellae, another important enzyme system which is driven by the proton motive force. It is apparent that the wheel has been in continuous use for at least 2000 million years.

BIOENERGETICS

Redox potentials: These provide a quantitative measure of oxidising or reducing power. Strong oxidising agents (like molecular oxygen) have positve redox potentials, good reducing agents have negative redox potentials. Electrons move spontaneously towards those compounds with the more positive redox potentials. The standard for the whole scale is pure hydrogen gas at 1 atmosphere pressure in contact with a platinised platinum electrode immersed in 1 molar acid. This combination is called a standard hydrogen electrode (SHE) and is defined to have a redox potential of zero.

Standard Redox Potentials (Eo) are measured with 50% oxidised form and 50% reduced form for the compound in question. [This is similar to pKa in the Henderson Hasselbach equation, which is equal to the pH value measured with 50% protonated buffer and 50% deprotonated buffer.] If

the oxidation / reduction reaction involves protons (many do) then you need 1 molar acid present as well. This is a bit inconvenient for biological systems, so we often work with Eo' which is the redox potential measured at pH 7. This has a major effect: at pH 7 hydrogen gas has a redox potential of -420 mV.

The effective redox potential depends on the proportion of the oxidised and reduced forms. This can make a big difference: the NADPH / NADP couple (maintained by cells almost 100% in the reduced form) is a much better biological reducing agent than NADH / NAD (no more than 30% reduced under normal conditions) despite the fact that their standard redox potenials are identical.

E = Eo + R T ln( [oxidised form] / [reduced form] ) / z F

(E is measured in volts, R is the gas constant [8.31 joules / degree / mole], T is the absolute temperature, F is the faraday constant [96,500 coulombs / mole], z is the number of electrons involved in the reaction and ln denotes the natural logarithm. If natural logs are replaced by base 10 logarithms, these must be multiplied by a conversion factor of 2.303)

Note that it is meaningless to speak of the redox potential for a single compound in isolation: both the oxidised and the reduced forms must be defined, although in some cases (e.g. NADH) the oxidised form (NAD) is obvious. For hydrogen gas the oxidised form could be either protons or water (you must specify which) while for oxygen the reduced form is usually either hydroxyl ions or water, but it could be superoxide or peroxide, and this would affect the value of the redox potential.

The numerical value of 2.303 R T / F is about 60mV at 37oC. Can you use this information to calculate the change in redox potential for hydrogen gas, going from 1 molar acid in a standard hydrogen electrode to the biological standard at pH 7.0 ?

A few redox potentials can be measured directly (by inserting a platinum electode into a mixture of the oxidised and reduced forms, and completing the circuit through a salt bridge connected to a suitable reference electrode) but biological redox potentials are more commonly obtained indirectly by studying the equilibrium position of reactions where one participant has a known redox potential. When everything is present in equimolar amounts, a good reducing couple can reduce a weaker couple. However, a weak reducing agent could reduce a stonger reducing agent, providing that the weaker couple were largely in the reduced form, and the stronger couple largely in the oxidised form. Similar considerations apply to oxidising agents, with the argument appropriately inverted.

When an oxidising reagent interacts with a reducing agent, the difference between their respective redox potentials E is related to the Gibbs free energy G for the overall reaction:

G = - z F E

(where G is measured in joules, E is measured in volts, F is the faraday constant [96,500 coulombs / mole] and z is the number of electrons transfered in the reaction). A little care is

needed with the arithmetical signs in deciding which number should be subtracted from what.

Remember that G is negative if the overall reaction is favourable.

The numerous electron carriers that make up the respiratory chain are arranged in the approximate order of their redox potentials: the best reducing couples at the substrate end and the best oxidising couples at the oxygen end. At key points along the chain, the difference in redox potential between adjacent carriers provides the driving force to pump protons out of the matrix space and into the cytosol as part of the overall energy coupling mechanism.

The redox potentials for some important biological reactions are listed in the table:

Chemical reaction Eo' (mV)

isocitrate => oxoglutarate + CO2 + 2e-1 -380

hydroxybutyrate => acetoacetate + 2e-1 -346

pyruvate + CoASH => acetyl-CoA + CO2 + 2e-1 ?

NADH => NAD+ + H+ + 2e-1 -320

lactate => pyruvate + 2e-1 -190

malate => oxaloacetate + 2e-1 -166

succinate => fumarate + 2e-1 +30

ubiquinol => ubiquinone + 2H+ + 2e-1 +45

cytochrome c2+ => cytochrome c3+ + e-1 +230

H2O => 1/2 O2 + 2H+ + 2e-1 +820

Nernst equation: This equation has various forms: do not be surprised if you find another version. The form most commonly encountered in biological systems relates the membrane potential to the concentrations of a diffusible ion in equilibrium with the potential on each side of the membrane:

= 2.303 R T log( [Cin] / [Cout] ) / z F

(where is the membrane potential in volts, R is the gas constant [8.31 joules / degree / mole], T is the absolute temperature, Cin and Cout are the two ionic concentrations, z is the electric charge on the ion, F is the faraday constant [96,500 coulombs / mole]. The factor of 2.303 arises from the use of log10 instead of natural logarithms.)

When the system reaches equilibrium, the tendency for the diffusible ion to escape through the membrane, down its concentration gradient, is exactly balanced by the electrical force attracting it in the opposite direction. The membrane potential changes sign if you reverse the orientation of the gradient, or if you substitute a diffusible anion for a diffusible cation keeping all the concentrations unchanged. Use your common sense to work this out: at equilibrium, the highest concentration of a positive ion will be on the negative side of the membrane, and vice versa.

At 37oC the value of 2.303 R T / z F is about 60mV so the membrane potential increases by 60mV for each tenfold increase in ion gradient. This is the same as the electrical output from a glass pH electrode (60mV per pH unit) which is not surprising because the voltage arises through the same mechanism.

Note that the ion must be able to cross the membrane with movement of charge for the above equation to apply, and it must have reached equilibrium with the electrical gradient. The Nernst equation does not apply to impermeant ions, or those which cross by an electroneutral exchange mechanism. No useful work can be obtained from an ion gradient subject to the Nernst equation (see below for a detailed discussion) but it is possible to obtain energy from an ion gradient which has NOT reached equilibrium, for example the sodium gradient across the plasmalemma, or the proton gradient across the mitochondrial inner membrane.

The Nernst equation can be used to calculate the mitochondrial membrane potential by measuring the distribution of a lipid soluble cation such as tetraphenylammonium, or rubidium plus valinomycin.

Gibbs free energy: ( G) This is the useful work which can be obtained from a chemical reaction, and reflects its displacement from equilibrium. No useful work can be obtained from a reaction which has reached equilibrium, and in this case G = 0.

For a chemical reaction where a series of reactants Rn form a series of products Pm:

R1 + R2 + R3 + ... <=> P1 + P2 + P3 + ...

the precise relationship between G and the extent of reaction is given by the Gibbs equation:

G = G0 + R T ln ( [P1] . [P2] . [P3] ... / [R1] . [R2] . [R3] ...)

(where R is the gas constant [8.31 joules / degree / mole], T is the absolute temperature, and ln( ) is the natural logarithm of all the product concentrations multiplied together, then divided by all the reactant concentrations multiplied together). If you prefer to work with base 10 logarithms, you must multiply the whole of the last term above [starting with R T ln( ....) ] by 2.303

G0 is the standard free energy for the reaction, measured when all the reactants and products are present at 1 molar concentration.

This graph shows the relationship between G and reaction progress for two different chemical reactions.

red reaction: G0 is positive

blue reaction: G0 is negative.

In both cases G is zero at equilibrium, but the red equilibrium position favours the reactants while the blue equilibrium favours the products.

The horizontal axis for this graph is R T ln ( [P1] . [P2] . [P3] ... / [R1] . [R2] . [R3] ...)

Since G is zero at equibrium, it follows that:

G0 = - R T ln ( [P1E] . [P2E] . [P3E] ... / [R1E] . [R2E] . [R3E] ...)

where the subscript E denotes the concentration of reactant or product present at equilibrium. Under these conditions, the terms inside the round brackets then represent the equilibrium constant, Keq for the overall reaction, leading to the important conclusion that:

G0 = - R T ln (Keq)

where Keq = [P1E] . [P2E] . [P3E] ... / [R1E] . [R2E] . [R3E] ...

Free energy of an ion gradient: No useful work can be obtained from ions subject to the Nernst equation (unless you alter the electrical gradient) because these ions have already reached equilibrium with the membrane potential, but energy can be obtained from the dissipation of a gradient for non-diffusible ions across the same membrane. There are two separable components to this free energy, one of which arises from the electrical gradient across the membrane, and the other from the difference in ionic concentrations. To calculate the free energy, imagine the ions performing some useful task as they escape across a tiny membrane separating two enormous reservoirs of effectively infinite capacity. Visualise moving one mole of the non-permeant ion from one side of the membrane to the other, keeping everything else (ionic concentrations, voltages) constant

G = z F + 2.303 R T log ( [Cout] / [Cin] )

(where G is the free energy, z is the charge on the ion, F is the faraday constant [96,500 coulombs / mole] and is the membrane potential, R is the gas constant [8.31 joules / degree / mole], T is the absolute temperature, Cin and Cout are the concentrations in the two compartments). The first term on the right hand side is like an electricity bill, basically amps x volts x time. The second term is a slightly modified form of the Gibbs equation, where the trapped ions are regarded as reactants, and the escaped ions are seen as products. G0 is zero

because no energy is available from the concentration term if the ion has the same concentration on both sides of the membrane. The factor of 2.303 allows for the use of base 10 logarithms in place of natural logs.

The above equation allows almost infinite opportunity for getting the signs muddled up. Remember that G is negative for favourable reactions. You need the correct sign for the membrane potential, and the correct charge on the ion, and remember that the second concentration term may either add to or subtract from the first electrical term, depending on whether Cout is greater or less than Cin. Use your common sense to visualise what is happening, and then insert the correct signs as appropriate.

Henderson Hasselbach equation: This relates the pH of a buffer solution to the ionisation constant for the buffer, and the proportion of the protonated and non-protonated forms:

pH = pKa + log10 ([deprotonated form] / [protonated form])

It is easy to remember this: the greater the proportion of the protonated form, the more acidic the buffer must be. For a weak acid buffer system such as acetic acid and sodium acetate, the free acetic acid is the protonated form and the deprotonated form is the salt. For an amine buffer, the salt would be the protonated form.

The pKa reflects the intrinsic affinity of the buffer for protons, and is simply log10 of the association constant, Ka:

BH       <=>      B-     +     H+ Ka = [BH] / ( [B-] . [H+] )

Again, it is easy to get this the right way round: strong bases have large pK values, an alkaline pH and a strong affinity for protons. When a buffer is exactly half-neutralised, the concentrations of the protonated and deprotonated forms are equal. At this point the system has maximum buffering capacity and the pH = pKa

MITOCHONDRIAL TRANSPORT SYSTEM

Normal operation of the respiratory chain creates both a pH differential and a voltage gradient of 30,000,000 volts/metre across the inner mitochondrial membrane. This demands highly specific transport proteins to control the movement of small ions across the membrane, and to prevent the dissipation of the various gradients.

The majority of inner membrane carriers are antiporters which exchange one molecule for another. If the electrical charges on the two molecules exactly balance the exchange will be an electroneutral process and the huge membrane potential will have no effect on the result. However, the pH gradient may affect the equilibrium position for an electroneutral exchange if one or more participants are acidic or basic.

Symports transport two molecules in the same direction. If the molecules have opposite charges this may still be an electroneutral process. It is experimentally difficult to distinguish between a proton symport and a hydroxyl antiport.

If the charges differ on the transported molecules for either symports or antiports, then there will be an electrical (sometimes called electrogenic) transport process. Such carriers can "feel" the membrane potential, which will exert a major influence on the result. In general the gradients affected by the pH component of the proton motive force are fairly modest, but those influenced by the membrane potential are substantial.

In a very few cases (e.g. for calcium uptake) the carrier simply allows the charged ion to traverse the membrane. This constitutes an electrical (or electrogenic) uniporter. The membrane potential exerts its full effect, leading to substantial concentration gradients if the reaction were able to reach equilibrium.

The principal transport systems are listed in the table below, which also shows some of the main inhibitors.

porter stoichiometry inhibitors comments

phosphate electroneutral exchange ofH2PO4

- for OH- NEMmersalyl

all mitochondria

dicarboxylate random electroneutral exchange ofmalate2- for succinate2- or HPO4

2-mersalyl malonate

all mitochondria

tricarboxylate electroneutral exchange of (citrate3- + H+ ) for malate2-

benzene 1,2,3

tricarboxylate

mammalian adipose tissueand liver: needed for fattyacid biosynthesis

oxoglutarate electroneutral exchange of malate2- for oxoglutarate2-

_ most mammalian tissuesneeded for the malateaspartate cycle

adenine nucleotides

electrical ATP4-/ADP3- exchange atractylosidebongkrekicacid

all mitochondria

glutamate electroneutral exchange ofglutamate- for OH-

_ mammalian liver: neededfor the urea cycle

aspartate electrical exchange of(glutamate- + H+ ) for aspartate-

_ most mammalian tissuesneeded for the malateaspartate cycle

calcium electrical uniport for Ca++ _ calcium uptake system

calcium electroneutral exchange ofCa++ for 2H+

_ mitochondrial calcium export system in liver

calcium / sodium

electroneutral exchange ofCa++ for 2Na+

_ mitochondrial calciumexport system in heart

sodium / proton

electroneutral exchange ofNa+ for H+

_ widely distributed

The charge imbalance associated with the adenine nucleotide carrier leads to a large difference in the ATP/ADP ratio between matrix space and cytosol. ATP is effectively "worth more" in the cytosol, because the ATP:ADP couple is maintained further away from equilibrium:

Cells do not, however, get something for nothing. The transport of ATP, ADP and phosphate across the inner mitochondrial membrane costs 33% additional energy, over the minimum required for the synthesis of ATP within the mitochondrial matrix compartment. This extra energy must be supplied by the respiratory chain. One additional proton is used to drive both of these transport systems: the positive charge on this single proton drives adenine nucleotide exchanger, while its acidity drives the phosphate uptake. The electrical and pH components of the proton motive force are exploited separately.

There has recently been great interest in the ATP:ADP carrier protein as a possible auto-antigen in dilated cardiomyopathy, a common and debilitating cardiac disease. This same enzyme is involved in the mitochondrial permeability transition and in apoptosis.

The high cytosolic ATP/ADP implies a very low cytosolic AMP concentration, as a result of the myokinase equilibrium. This enables 5' AMP to serve as an emergency signal, which indicates a threat to the ATP supply. Several key enzymes (notably glycogen phosphorylase and phospho- fructokinase) are strongly activated by low concentrations of 5'AMP. This nucleotide also gives rise to adenosine which stimulates blood flow to active tissues. Note that 5'AMP differs from 3'5' cyclic AMP produced by adenyl cyclase.

Muscle contraction apparently requires a very high ATP/ADP ratio, and it is difficult to maintain a low myofibrillar ADP because it will not diffuse quickly enough back to the mitochondria at low ADP levels. (Diffusion rate is directly proportional to metabolite concentration.) Active muscles contain creatine phosphokinase, (CPK) and large amounts of creatine and creatine phosphate. These compounds equilibrate with the adenine nucleotide pools. It is thought that the high concentrations of these highly diffusible energy carriers increase the maximum energy transport rate.

Gene knockout experiments have shown that mice lacking both the mitochondrial and myofibrillar CPK iso-enzymes exhibit abnormal development, however mutants deficient in only one iso-enzyme are superficially healthy. It is difficult to see why both CPK genes should be widely retained if they don't do anything. It remains to be established whether the single mutants can maintain the sustained work outputs achieved by the wild-type controls.

One of the most important asymmetric transporters is the aspartate carrier, which in mammals plays a key role in the re-oxidation of glycolytic NADH by the malate-aspartate cycle. This

cycle is necessary because the inner membrane is not permeable to either NAD or NADH. The component reactions must revolve twice for each molecule of glucose oxidised by the cell. The two enzymes involved, malate dehydrogenase (MDH) and glutamate oxaloacetate transaminase (GOT) are among the most active in the body. Both enzymes exist in mitochondrial and cytosolic variants, but all four proteins are coded by nuclear genes.

Aspartate- swops for glutamate plus a proton, so the full proton motive force is applied to the aspartate porter. The overall effect is to bias this otherwise symmetrical cycle, and maintain the cytosolic compartment in a relatively "oxidising" state (with a low NADH/NAD ratio) while the mitochondrial compartment is kept correspondingly reduced. This arrangement suppresses lactate formation during aerobic glycolysis. Invertebrate species employ alternative shuttles to achieve the same effect, and it is doubtful if eukaryotic cells could work at all without some such arrangement.

It is important to realise that no porter means no transport. Oxaloacetate and fumarate, for example, bind very badly to the dicarboxylate carrier, and are not transported at any significant rate. If both malate and oxaloacetate were transported it would be impossible to maintain the redox differential for NADH / NAD which exists between the mitochondrial matrix space and the cytosol.

Metabolite porter genes are located in the nucleus and are only expressed in those tissues which require them. For example, liver mitochondria have porters for ornithine and citrulline, which are required for the urea cycle, but these are not found in other tissues. The citrate carrier is found only in liver cells and adipocytes, where it is needed for lipogenesis

The outcome of many substrate oxidation experiments in vitro is determined by the porter specificity. For example: most animal mitochondria oxidise succinate almost quantitatively to malate, because neither fumarate nor oxaloacetate can be transported out of the matrix space and there is no source of acetyl-CoA to produce citrate.

Even in liver mitochondria, which possess a glutamate / hydroxyl antiporter, glutamate added alone mainly yields aspartate via glutamate oxaloacetate transaminase (GOT), oxoglutarate dehydrogenase (OGDH), succinate thiokinase (STK), succinate dehydrogenase (SDH), fumarase (FUM), malate dehydrogenase (MDH) and the aspartate porter.

However, if glutamate and malate are added together, mammalian mitochondria initially produce equal amounts of aspartate and oxoglutarate using the mitochondrial half of the malate - aspartate cycle. The aspartate and oxoglutarate porters, GOT, MDH and the respiratory chain are the only enzymes involved under these conditions.

Self assessment question: If mitochondria were incubated with oxoglutarate alone, which pathways would be operative and which compound(s) would be formed?

In some cases, e.g. for calcium ions, an electrical carrier is used for uptake and an electroneutral system is used for export. The two components of the proton motive force are used to drive the ion in opposite directions. This may be exploited by cells to regulate intra-mitochondrial calcium to a different value to the cytosolic concentration.

Many important metabolites show an asymmetric distribution across the mitochondrial inner membrane. The operation of the electroneutral anion carriers leads to a modest accumulation of polybasic acids (especially citrate) within the matrix space. In calculating the equilibrium distribution, it is only the "bottom line" which counts - the net number of protons which crossed the membrane by whatever process in order to accumulate the anion. The detailed mechanism is irrelevant, and any intermediate "swops" are discounted. If the overall reaction for electroneutral metabolite accumulation is:

anionn-(out) + nH+

(out) <=> anionn-(in) + nH+

(in)

then simple mass action considerations lead to the conclusion that:

[anionn-in ] / [anionn-

out ] = ([H+out] / [H+

in])n

This is helpful for the operation of the citric acid cycle, and also has implications for the regulation of glycolysis and lipogenesis by phosphofructokinase and acetyl CoA carboxylase, where citrate is an allosteric effector.

Mitochondrial swelling experiments: If it is desired to study membrane transport in isolation it is often necessary to block further metabolism with cyanide or rotenone. The most accurate studies of transport kinetics involve the rapid separation of mitochondria from their incubation medium using ultrafiltration or centrifugation through silicone oil. These complex and time

consuming experiments require radioactive tracers to correct for "carry over" of the incubation medium into the mitochondrial fraction. Large scale metabolite movements will, however, produce alterations in matrix volume through osmotic mechanisms. A qualitative estimate of transport rates can thus be obtained by measuring the changes in light scattering from a turbid mitochondrial suspension.

These results were obtained from 0.5 mg liver mitochondria suspended at pH 7 in a spectrophotometer cuvette with 2.5 ml of 150 mM potassium acetate. Acetate ions can penetrate rapidly by an electroneutral process as free acetic acid, but no swelling is observed initially because the potassium cannot enter. Addition of valinomycin has no effect because this ionophore catalyses electrical movement. Nigericin allows an electroneutral exchange of potassium ions for protons and rapid swelling takes place.

Passive swelling in isotonic ammonium salt solutions can be used to study the major anion transport systems in cyanide-blocked mitochondria. The solutes move passively down their electrochemical gradients. Ammonium ions penetrate rapidly as free ammonia without movement of charge, and swelling is observed if the anion is also transported by an electroneutral process. It can be shown that phosphate, malate, succinate, oxoglutarate, citrate and sodium are all taken up by electroneutral mechanisms, and the phosphate / hydroxyl, phosphate / dicarboxylate, dicarboxylate / tricarboxylate and malate / oxoglutarate exchangers can be demonstrated.

It is also possible to observe "energised swelling" using intact mitochondria where solutes are accumulated against a concentration gradient using energy from respiration or from external ATP. There are substantial differences between the energised and passive processes, but in every case (except for ATP-driven pumps) net ion movements are always down the electro-chemical gradient.

ORIGIN AND EVOLUTION OF MITOCHONDRIA

The earth was formed about 4,500,000,000 years ago. The original atmosphere was a reducing one, containing (among other constituents) nitrogen, methane, carbon dioxide, hydrogen sulphide and water vapour. There was no free oxygen, so oxidative phosphorylation was not an option for the earliest organisms, which relied instead on a fermentative metabolism. Pre-biotic synthesis in thunderstorms and volcanic vents is believed to have left a rich legacy of the most common stable metabolites, which were scavenged by our distant ancestors.

The earliest living organisms were subject from the start to a relentless selection pressure for better substrate uptake systems. It is conceivable that trans-membrane proton gradients were originally used to drive substrate uptake. The F1ATPase may at first have fulfilled this important

function, as the sodium gradient and the Na/K-ATPase still do today. Selection pressures would favour efficient ion pumping, leakproof membranes and steadily increasing gradients in order to scour the remaining metabolites from a depleted environment.

The importance of substrate uptake is that even marginal improvements yield an immediate selective advantage. In this situation the evolution of the earliest "photosynthesis" does not appear an unduly difficult step, serving initially to supplement pre-existing ion gradients from a widely available free power supply.

The earliest photosynthetic organisms were unable to produce oxygen from water, and relied instead on a variety of easier electron donors such as hydrogen sulfide and ferrous iron. They titrated the earth's crust, exhausting one reductant after another, subject always to the constant pressure to handle stronger oxidants and bigger gradients. It is likely that photosynthetic electron transport and oxidative electron transport systems evolved together, starting in each case from the most highly reducing end of their respective electron transport chains.

The transhydrogenase may be the most ancient part of the respiratory chain, capable of energising the outer membrane in a primitive fermentative bacterium long before any oxygen was present in the earth's atmosphere. The other respiratory chain components were probably added sequentially, as more effective oxidants slowly became available over hundreds of millions of years through the photosynthetic activities of the cyanobacteria and, very much later, the green plants. Meanwhile the selection pressure continued for larger and larger ion gradients. Eventually the trans-membrane pH gradient was sufficient to force the F1ATPase backwards (in its modern physiological direction) and modern photosynthesis and oxidative phosphorylation became possible.

All the gaseous oxygen in the present atmosphere is believed to have had a biological origin, and was mostly formed sometime between 3,000,000,000 and 1,000,000,000 years ago, as a result of photosynthesis by cyanobacteria and the earliest green plants. Electron transport was a prokaryotic invention, and its practitioners must have enjoyed a tremendous selective advantage from efficient ATP synthesis. Moreover, the rising atmospheric concentration of highly toxic and reactive oxygen was a serious threat to our eukaryotic ancestors. Even today, despite the evolution of effective anti-oxidants, most of our tissues maintain their intracellular oxygen concentration in the micromolar range,1000 times below the bloodstream value.

Primitive eukaryotes, however, had evolved one technique which no prokaryote could perform: they were sufficiently large and flexible to swallow other organisms whole, probably digesting them for food. How much more efficient, though, to keep a few aerobic bacteria as guests, supplying them with substrates in return for an endless supply of ATP? The original bacterial ion porters could continue to operate, and a single host cell mutation might be sufficient to insert an adenine nucleotide carrier through the bacterial cell wall.

Mitochondria are thought to have evolved at least 2000 million years ago from primitive bacteria which enjoyed such a symbiotic relationship with early eukaryotic cells.

It must have been difficult initially to synchronise the activities of the two genomes, and there followed a gradual transfer of mitochondrial genetic functions to the eukaryotic cell nucleus, where they were better integrated with the other cellular controls. This process has progressed to varying extents in different species, so that yeast and mammalian mitochondria differ slightly in the functions which they have retained.

Mitochondria still show some signs of their ancient origin. Mitochondrial ribosomes are the 70S (bacterial) type, in contrast to the 80S ribosomes found elsewhere in the cell. As in prokaryotes there is a very high proportion of coding DNA, and an absence of repeats. Mitochondrial genes are transcribed as multigenic transcripts which are cleaved and polyadenylated to yield mature mRNAs. Unlike their nuclear cousins, mitochondrial genes are small, generally lacking introns, and the chromosomes are circular, conforming to the bacterial pattern.

In addition to the mitochondrial ribosomes and transfer RNA, the small amount of mitochondrial DNA codes for only 13 polypeptides in humans. These are mainly the hydrophobic cores of the major trans-membrane proton pumps, which are sticky and insoluble and difficult to move around the cell. All the remaining mitochondrial genes have migrated to the nucleus, and the hundreds of other mitochondrial proteins are now imported from the cytosol.

Maternal inheritance: The vast majority of mitochondria in a fertilised egg derive from the hundreds of thousands of mitochondria in the large maternal egg rather than the small number in the tiny sperm. As a result most mitochondrial traits appear to be transmitted exclusively through the maternal line.

Nevertheless, there are obvious difficulties with this hypothesis. If maternal transmission were the only mechanism, and there were no device within each cell to eliminate defects and synchronise the multiple copies of the mitochondrial DNA, within a relatively short period there would be thousands of mitochondrial variants. These are not observed in practice, despite the fact that mitochondrial DNA is less well protected than nuclear DNA and mitochondrial DNA replication lacks some of the error detection systems employed in the cell nucleus. Single-parent inheritance also denies to a species the evolutionary advantages of recombination and sexual reproduction.

Electron micrographs show that sperm contain two types of mitochondria: the large spiral structure which provides the power for flagellar movements, and two smaller, less specialised mitochondria packaged next to the male pronucleus with the paternal DNA. Whatever the function of this curious arrangement, its effects are not apparently manifest in the immediately succeeding generation.

Mitochondrial Eve: Assuming a largely maternal inheritance, analysis of human mitochondrial DNA is consistent with the hypothesis that the entire modern human race is descended from a single female or a group of closely related sisters, who lived in Africa about 200,000 years ago.

There are numerous objectors to this hypothesis, nevertheless the limited variability of our nuclear DNA also suggests that our ancestors recently went through an "evolutionary bottleneck"

with a very small number of survivors. Our presence on the planet appears to have been a close run thing.

BIOGENESIS OF MITOCHONDRIA

The human mitochondrial genome is only 16.6kb and among the smallest in the animal kingdom. The circular chromosome was completely sequenced by Sanger's team in 1981 and the map is shown below:

The heavy H strand has a higher guanine content, and in this diagram is transcribed in a clockwise direction as a single RNA molecule starting from the PH promoter. The light L strand is transcribed anticlockwise from the PL promoter. Both RNA transcripts are later cleaved to yield functional RNA molecules. Replication of the heavy strand by DNA polymerase commences anticlockwise from the OH replication origin; this eventually exposes the OL origin allowing replication of the light strand to be completed. The red-coloured region near the promoters is known as the D-loop and contains a short length of triple stranded DNA.

The mitochondrial genetic information is very densely packed. There are no introns and only tiny gaps between the genes. [In the diagram above, N1 - N6 are NADH dehydrogenase subunits, cyt b is cytochrome b, OX1 - OX3 are cytochrome oxidase subunits, and the genes for two ATPase subunits partly overlap.] There are 37 mitochondrial genes in total, but only 13 of these code for polypeptides, the remainder being the 2 ribosomal subunits and 22 types of transfer RNA. All the hundreds of other mitochondrial proteins, including DNA polymerase, RNA polymerase, amino acid activating enzymes and all the ribosomal proteins are coded by nuclear genes and imported from the cytosol.

There are about 2000 copies of the mitochondrial genome in a typical cell, so that despite its small size it comprises about 0.5% of the DNA mass. Mitochondrial DNA replication appears to be less accurate than nuclear copying, and it is far from clear how consistency is maintained between these multiple copies, or how their replication is synchronised with the remainder of the cell.

There are substantial differences between the proteins present in mitochondria from different tissues, reflecting the tissue specific patterns of nuclear gene expression. Protein turnover, however, seems to be fairly slow and mitochondrial protein composition does not respond very quickly to dietary or hormonal stimuli. The total number of mitochondria per cell can be changed (for example, through muscle activity) over the course of several weeks.

The import of cytosolic proteins to the mitochondrial matrix space takes place at specialised sites where the inner and outer membranes are in close contact. Cytosolic precursors are marked for import with an N-terminal leader sequence, bearing a substantial positive charge. The leader folds to form an amphipathic alpha helix where the hydrophobic amino acids are concentrated along one face. Receptor proteins in the outer membrane recognise sub-sets of these import signals, and help to direct the precurors towards the import channel. Mitochondrial proteins are usually more basic than their cytosolic counterparts. They probably have a net positive charge at physiological pH, and are strongly influenced by the membrane potential. It appears that the proton motive force provides part of the energy for protein uptake.

Proteins pass through the membranes in an extended linear configuration. It is important that they should not fold fully before import, since the correctly folded enzymes could not pass through the hole. Folding is temporarily prevented in the cytosol by binding to the chaperone protein hsp70, while two further mitochondrial chaperones hsp60 and hsp70 supervise re-folding of the imported proteins after they have entered the matrix space. ATP hydrolysis is required in the cytosol and in the mitochondria for successful import of functional proteins, in addition to the membrane potential and pH gradient. This ATP requirement is probably associated with protein folding.

The matrix targeting sequence is cleaved from the imported proteins by a matrix protease. If the imported protein is destined for the intermembrane space (e.g. myokinase) cleavage of the first signal sequence is believed to expose a second signal directing re-export of the protein through the inner membrane. (The unusually small protein apo-cytochrome c apparently uses the non-specific outer membrane channels to reach the inter-membrane space. Chelation of heme, and consequent adoption of the native protein conformation locks this particular protein into its final position.)

MITOCHONDRIAL DISEASES

Primary mitochondrial diseases are relatively rare, probably because major defects in the Krebs cycle or the respiratory chain are incompatible with life and many affected embryos die at an early stage. Nevertheless, about 150 different types of hereditary mitochondrial defect have been reported, and mitochondria play an important part in several common conditions. Mitochondrial DNA is maternally inherited through the egg cell cytoplasm, but many of the inherited defects map to the nuclear genome since the majority of mitochondrial proteins are imported from the cytosol. Heteroplasmy is a complicating factor: affected cells may contain a mixed mitochondrial population, and the defect may only involve a proportion of the mitochondria. (In healthy people all the copies of the mitochondrial DNA are identical.) Mitochondrial defects may be confined to a limited range of tissues, and, unexpectedly, they may change substantially as the patient ages.

There has recently been great interest in mitochondrial diseases with an autoimmune component, and in those involving apoptosis (programmed cell death).

Respiratory defects: A huge variety of individual defects have been described, affecting one or more of the respiratory chain redox carriers. (A defect in mitochondrial protein import, for example, might affect dozens of enzymes to a greater or lesser extent.) The tissues which rely most extensively on aerobic metabolism are most severely affected, so patients commonly present with a myopathy or encephalopathy, or both. Lactic acidosis, muscular weakness, deafness, blindness, ataxia and dementia are common findings. These mitopathies are often fresh mutations, so there is no family history. In many cases the diseases are obvious from birth, but may develop in later life if the number of defective mitochondria increases with age.

Light microscopy of muscle biopsies frequently reveals a proportion of "ragged red" fibres or the absence of key respiratory enzymes from particular cell types after histochemical staining. Abnormal mitochondria may be visible in the electron microscope: for example "parking lot" mitochondria where the normal cristae are replaced by a rectangular grid. Even where the nature of the mutation has been identified by DNA sequencing, there is only limited correlation between the genetic defect and the time course of the disease or the severity of the symptoms.

The classification of this heterogeneous group of diseases leaves much to be desired, but the following are typical examples: myoclonic epilepsy with ragged red fibres (MERRF), mitochondrial encephalopathy with lactic acidosis and stroke (MELAS), neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP).

Auto-immune diseases: There are some relatively common diseases where patients have been reported to produce auto-antibodies against mitochondrial proteins. These include primary biliary cirrhosis (pyruvate dehydrogenase complex), dilated cardiomyopathy (adenine nucleotide porter) and Leber's heriditary optic neuropathy (LHON).

LHON is the most curious, because the antigen displayed on the plasmalemma is derived from a mitochondrially-encoded subunit of NADH dehydrogenase (complex 1). LHON is closely related to multiple sclerosis, and is the most common cause of blindness in otherwise healthy young men. It mainly affects young adult males, but it is maternally transmitted and female relatives are often carriers. It can be distinguished from an X-linked recessive condition, because male patients are normally unable to transmit the mutation to their children.

The permeability transition: Under conditions of extreme stress (e.g. pathological calcium ion concentrations, free radical mediated oxygen damage) mitochondria undergo an autocatalytic collapse, associated with the loss of the normal membrane potential and a complete failure of ATP production. The transition is irreversible and is normally a prelude to cell death. It is routinely prevented during the laboratory isolation of mitochondria by including a calcium ion chelator (EDTA or EGTA) in the isotonic preparation medium.

The transition involves the incorporation of subunits from the adenine nucleotide porter, and other proteins derived from the outer membrane, into a large pore which allows unrestricted access of small ions to the mitochondrial interior. Pore formation is favoured by atractyloside

and inhibited by bongkrekic acid, and can also be blocked by the fungal toxin cyclosporin A which binds to a protein called cyclophillin in the mitochondrial matrix. [Cyclosporin A is a cyclic peptide which is widely used as an immuno - suppressant after transplant surgery, but this may be unconnected with its effects on mitochondria.]

Apoptosis: The mitochondrial permeability transition is believed to be involved in the suicidal process of apoptosis, or programmed cell death. This elaborate self-destruction cascade is responsible for the programmed loss of cells during tissue differentiation, the self-destruction of tumours and virally - infected cells, and the unwanted cell damage which follows loss of tissue perfusion in cardiovascular disease. It is proposed that mitochondria which undergo the permeability transition release a protease from the inter-membrane space which then activates the subsequent nuclear stages of the apoptotic cascade.

Apoptosis is closely regulated at numerous points along the cascade. Key effectors include (1) the tumour suppressor protein p53, which induces cells to undergo apoptosis when irreparable DNA damage is detected, and (2) the proto-oncogene bcl-2 which prevents the permeability transition, suppresses apoptosis and potentially allows the survival of damaged or cancerous cells. Bcl-2 is concentrated in the mitochondrial outer membrane, where it is closely involved in regulating the permeability transition.

KEY POINTS TO REMEMBER

The key points are as follows:

1) Mitochondria are subcellular organelles containing the Krebs cycle, fat oxidation pathway and the respiratory chain, which produce almost all of the 70kg of ATP used each day in the human body. They can be purified from tissue homogenates by differential centrifugation, using non-penetrant isotonic media to provide osmotic support. A few tissues (e.g. red blood cells, eye lens) have no mitochondria, and cannot respire aerobically.

2) Mitochondrial respiration can be measured with an oxygen electrode. Similar equipment is used in blood gas analysers, pollution monitoring and industrial process control. How does it work, and what are the principal sources of error?

3) Understand precisely what is meant by the respiratory control index, P:O ratio and uncoupling. Calculate P:O ratios for succinate, and for glutamate + malate using intact, coupled mitochondria.

4) Several of the mitochondrial components undergo spectral changes on oxidation and reduction. These observations suggest that a series of redox carriers (arranged approximately in the order of their oxidation/reduction potentials) transport reducing equivalents (electrons or

hydrogen atoms) from substrates to oxygen. This sequence of carriers is known as the respiratory chain.

5) Understand the use of specific inhibitors to delineate biochemical mechanisms. Appreciate the differences between respiratory chain inhibitors, phosphorylation inhibitors and transport inhibitors using cyanide, antimycin, rotenone, TTFA, oligomycin, mersalyl and atractyloside as examples.

6) Energy captured by the respiratory chain is not converted directly into ATP. There is instead a temporary "clearing house" known as the high energy pool. Its chemical nature was a mystery for many years, but now we know that it is an electrochemical gradient for hydrogen ions across the inner mitochondrial membrane. In human mitochondria this ion gradient or proton motive force has a large electrical component and a small pH component.

7) Understand the operation of the major transmembrane ion pumps and the ATP synthetase.

8) Lipid bilayers are exceedingly impermeable to small ions, including protons and hydroxyl ions. Transmembrane ion gradients are another form of energy, interconvertible with chemical, electrical and mechanical energy. The chemiosmotic mechanism is universal in living organisms and is also exploited by chloroplasts for photosynthesis, and by bacteria.

9) Understand the mechanism of ionophores, the distinction between electroneutral and electrical carrier mechanisms, and the importance of trans-membrane ion and potential gradients. Know the meaning of symports, antiports and electrogenic uniports. Understand where the Nernst equation applies.

10) Appreciate the use of light scattering as a crude measure of particle size and the use of passive swelling in isotonic ammonium salt solutions to demonstrate the principal transport sytems. Study the interactions between the carriers and explain the effect of phosphate on malate uptake. Why does passive swelling in sodium (but not potassium) phosphate indicate the existence of a sodium/proton anti-porter?

11) Understand why the oxidation products from individual substrates are constrained by the transport systems in the inner mitochondrial membrane, and by the availability of other co-substrates, such as acetyl-CoA.

12) The electroneutral uptake systems for small anions and cations produce relatively modest concentration gradients since they exploit only the pH component from the proton motive force, but the electrical adenine nucleotide carrier produces a large gradient for ATP since it can exploit the much larger electrical differential across the mitochondrial inner membrane.

13) The Gibbs free energy G available from a physical process or chemical reaction depends on the distance away from equilibrium. This means that ATP has a higher free energy in the cytosol than it does in the mitochondria, and this is important for many cellular activities.

14) Many metabolites are unevenly distributed across the mitochondrial membrane. Energy driven aspartate / glutamate exchange is important for keeping the cytosol highly oxidising, and preventing the aerobic synthesis of lactic acid.

15) Mitochondria probably evolved from symbiotic bacteria. They still contain a small amount of DNA, which codes for 70S (bacterial type) ribosomes, transfer RNAs and thirteen polypeptides. These are mostly the intensely hydrophobic cores of the major proton pumps. Understand the mechanism whereby hundreds of other mitochondrial proteins are imported from the cytosol. Mitochondrial DNA appears to be maternally inherited.

16) Mitochondria are involved in many disease processes, although some defects may not be detected as a result of heteroplasmy, or because the affected embryos are not viable. There is good evidence for auto-immune effects, and the mitochondrial permeability transition is thought to play an important part in apoptosis.

ASSIGNED TOPICS

Group of Jul 15 Jeff Rxns of glycolysis (energy yield), alternate fates of pyruvate,

hormonal regulation of glycolysis; Clinical cases related to

glycolysis;

Jul 15 Jashper rxns, substrates and regulations of gluconeogensis; rxns of

CAC; stoichiometry of CAC and regulation;HMP rxns

July 15/22 Krizzia Uses of NADPH; G6PD;Fructose, lactose and galactose

Metabolism; structure and function of glycogen

July 22 Angelica Synthesis of glycogen and its regulation; degradation of

glycogen and its regulation;Clinical problems related to g

lycogen metabolism; Synthesis and degradation of glycosamine

glycans

July 22/29 Simel Structure of FA’s; de novo synthesis of fa’s; mobilization of

stored fats and oxidation of fa’s; PG’s and related compounds

July 29 Wilson Structure, synthesis and degradation of phospholipids; structure,

synthesis and degradation of glycosphingolipids

July 29 Czarina cholesterol and steroid metabolism

ASSIGNED TOPICS

Group of Jul 15 Irish Rxns of glycolysis (energy yield), alternate fates of pyruvate,

hormonal regulation of glycolysis; Clinical cases related to

glycolysis;

Jul 15 Irish and Fran rxns, substrates and regulations of gluconeogensis; rxns of

CAC; stoichiometry of CAC and regulation;HMP rxns

July 15/22 Fran Uses of NADPH; G6PD;Fructose, lactose and galactose

Metabolism; structure and function of glycogen

July 22 Jamaica Synthesis of glycogen and its regulation; degradation of

glycogen and its regulation;Clinical problems related to g

lycogen metabolism; Synthesis and degradation of glycosamine

glycans

July 22/29 Jamaica and Jean Structure of FA’s; de novo synthesis of fa’s; mobilization

of stored fats and oxidation of fa’s; PG’s and related compounds

July 29 Jean Structure, synthesis and degradation of phospholipids; structure,

synthesis and degradation of glycosphingolipids

July 29 Myzy cholesterol and steroid metabolism

FOR EVERYONE: Every group shall submit their digested notes* on topics that will be reported on the coming session a week prior to that session. You may submit handwritten notes. This practice will somehow develop discipline on reading, self directed and cooperative learning. Synthesis and enhancement activities will be given during the session. Do your visual aids in any manner you wish. Just make sure the visuals will help facilitate your report. Reports need not be rigid and classic. You may do creative reporting.

*notes from group discussions of the topic after reading; summary (digested=absorbed) notes