succinate dehydrogenase fast myosin immunofluorescence myosin ATPase activity
Use your lecture notes and biochemistry textbooks to help you answer questions 1-6 and also complete Figure 1, using the information from Table 1, before coming to the session. You may find it useful to look at Human Metabolism (R. Bronk), Marks' Basic Medical Biochemistry (Smith, Marks & Lieberman) or Biochemistry (Mathews, Van Holde & Ahern) for completing Figure 1.
There are 2 Powerpoint presentations on the VLE which may help with your preparation for this work session.
Bring a biochemistry textbook to the session
Metabolism is a complex interconnecting network of metabolic pathways. Some sequences of reactions are required to break down and oxidise metabolites in order to produce ATP - the 'energy currency' of the body, whilst others use the chemical energy from ATP for functions such as the synthesis of new compounds, the maintenance of ion balances across membranes and for performing physical movements in contractile tissues.
A consequence of these metabolic processes is the production of heat, and in warm blooded animals it is crucial that appropriate mechanisms exist to tightly regulate body temperature. Without such mechanisms, metabolic processes, relying on enzymes whose rate of reaction is optimal at a particular body temperature, will not operate efficiently. Heat exchange occurs between the interior of the body and the skin, and its regulation depends largely upon changes in blood flow to the skin. Heat brought by blood to the skin can be lost via conduction, convection, radiation and evaporation. Dilation of the blood vessels in the skin allows more heat to be lost, whilst contraction of the surface vessels reduces heat loss.
At low temperatures, the body must be able to generate internal heat to survive. Shivering is a mechanism by which repetitive muscle tremor generates extra internal heat. In contrast to this, many animals, (especially those that hibernate) and also human neonates have a specialised tissue called brown adipose tissue that is also able to produce heat, in a process known as'non-shivering thermogenesis'. The mitochondria in this tissue contain a protein called thermogenin which can form pores through the inner mitochondrial membrane, thus acting as an uncoupling agent (the significance of uncouplers is discussed later in this Work Session).
At high external temperature or when the body needs to dissipate heat produced internally from metabolism and muscular exercise, efficient cooling systems must be activated. By simply considering the energy content of food consumed by an average human male, and ignoring any other heat losses from the body, if a daily intake of 12,000MJ was instantaneously converted into heat, it would cause a rise in body temperature of 48C! In adult humans, muscle comprises a large proportion of the body by weight and its metabolism accounts for most of our enenrgy expenditure. As such, it also accounts for much of the rise in body temperature experienced after exercise. If cooling systems were not functioning, it can be calculated that a healthy adult human exercising at 50,000J per minute, with 75% of heat being dissipated internally and 25% delivered to the outside as physical work, would die within around 40 minutes from a fatal rise in temperature of 4C.
Humans employ the evaporation of sweat from the skin in order to lose excess heat, and in the first hypothetical example above, disregarding other means of losing heat, the 48C temperature would require nearly 5 litres of sweat to maintain the body at the correct temperature.
Skeletal muscle fibres are classified into three main categories, 1, 2A and 2B differing in colour (red / white) and in contraction speed:
succinate dehydrogenase fast myosin immunofluorescence myosin ATPase activity
The three consecutive serial sections above were stained to show mitochondrial enzymes on the left, myosin isoforms by direct immunofluorescence in the centre and the rate of ATP hydrolysis by myosin on the right, where a darker stain means more activity. Identify the principal fibre types:
Type 1 or slow oxidative fibres have a slow contraction speed and a low myosin ATPase activity. These cells are specialised for steady, continuous activity and are highly resistant to fatigue. Their motor neurones are often active, with a low firing frequency. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic metabolism and prefer to use fat as a source of energy, which is a more economical fuel to use than glucose. These are the marathon runner's muscle fibres.
Type 2A or fast oxidative-glycolytic fibres have a fast contraction speed and a high myosin ATPase activity. They are progressively recruited when additional effort is required, but are still very resistant to fatigue. Their motor neurones show bursts of intermittent activity. Again, these cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic metabolism and can use either glucose or fats as a source of energy. These are general purpose muscle fibres which give the edge in athletic performance, but they are more expensive to operate than type 1.
Type 2B or fast glycolytic fibres have a fast contraction speed and a high myosin ATPase activity. They are only recruited for brief maximal efforts and are easily fatigued. Their motor neurones transmit occasional bursts of very high frequency impulses. These are large cells with a poor surface to volume ratio and their limited capillary supply slows the delivery of oxygen and removal of waste products. They have few mitochondria and little myoglobin, resulting in a white colour (e.g. chicken breast). They generate ATP by the anaerobic fermentation of glucose to lactic acid (and are consequently the least economical to use). These are sprinter's muscle fibres, and are of no use for sustained performance.
Type 1 and 2 muscles have different preferences for metabolic fuels, what are they?
What is the preferred metabolic fuel for heart?
What are the consequences of a high level of lactic acid in muscle?
What is the metabolic role of carnitine?
What symptoms would you expect patients deficient in carnitine to experience?
In which tissues (excluding type I muscle) would you expect to find many mitochondria?
How would (1) heat production and (2) oxygen consumption of these tissues compare to tissues with fewer mitochondria?
A schematic diagram of the components of the electron transport chain within the inner mitochondrial membrane is shown in Fig 1. Label the components appropriately using information from Table 1. Also indicate on the diagram where the reducing equivalents enter the chain (from NADH, NADPH and FADH2), the direction of movement of electrons and protons, the sites of oxygen reduction and ATP synthesis. In addition, label the transporters that carry phosphate, ADP and protons into the matrix.
Table 1. Some components of the oxidative phosphorylation system
| Component | Subcellular location | Function |
Complex 1 | membrane spanning multi-subunit protein | proton pump |
| Complex II | matrix side of inner membrane | insufficient DG to pump protons |
ubiquinone [co-enzyme] | inner membrane lipid phase | mobile carrier |
| Complex III | membrane spanning multi-subunit protein | proton pump |
cytochrome c | inter-membrane space | mobile carrier |
| Complex IV | membrane spanning multi-subunit protein | proton pump |
| F0/F1ATPase | membrane spanning multi-subunit protein | freely reversible proton pump |
| adenine nucleotide and phosphate transporters | membrane-spanning proteins | import/export ATP, ADP and Pi |
The best guess at present is that complex I pumps four protons (and hence) four electrical charges from the matrix space to the cytosol, while complexes III and IV together shift six protons (and six charges) for each pair of electrons traversing the electron transport chain. This leaves the matrix space about 0.5 pH units alkaline and 150mV negative with respect to the extra-mitochondrial compartment. These gradients together constitute the proton motive force.
Many dehydrogenases (DHs) e.g. pyruvate DH, lactate DH, malate DH, produce NADH as a product. This can transfer reducing equivalents (electrons) to Complex I, which then passes electrons to ubiquinone, simultaneously pumping protons into the intermembrane space.
A small group of dehydrogenases including succinate dehydrogenase (an enzyme in the citric acid cycle) produce FADH2 rather than NADH. FADH2 can pass reducing equivalents to ComplexII and then on to ubiquinone without any proton pumping.
F0/F1ATPase is also known as ATP synthase. It is a freely-reversible rotary proton pump which readily switches direction, depending on the magnitude of the proton motive force. Nine protons are required to drive a complete rotation, leading to the manufacture of three molecules of ATP. It catalyses the interconverion of ATP and ADP plus inorganic phosphate in the matrix compartment. These substrates and products must be imported / exported to the cytosol in separate steps, which consume a fourth proton for each completed ATP.
Figure 1. Arrangement of electron transport components in the inner mitochondrial membrane
IntermembraneMatrix
Space
INHIBITORS OF OXIDATIVE PHOSPHORYLATION
Our understanding of the electron transport chain has been increased by using compounds which inhibit one or more components of the system, and consequently it has been possible to distinguish the electron transport system from the phosphorylation system.
Whilst the passage of electrons down the electron transport chain is associated with the consumption of oxygen by a cell (where inhibition of one process results in inhibition of the other), this is only linked to the production of ATP when H+ can travel back into the matrix through the ATPase.
Some compounds called uncouplers are able to dissociate these two processes by creating a 'short circuit' back across the inner membrane.
The best-studied inhibitors of electron transport components and ATP synthesis are listed below.
Rotenone inhibits Complex I in the respiratory chain but does not stop electrons feeding into the electron transport chain at complexes II,III and IV.
Thenoyl trifluoroacetone (TTFA) blocks the respiratory chain at complex II.
Antimycin A blocks the respiratory chain at complex III.
Cyanide blocks Complex IV and prevents respiration with all substrates that feed electrons into the electron transport chain.
Oligomycin blocks ATP synthesis (and degradation) by the F1ATPase, so it abolishes ADP-stimulated respiration in intact mitochondria.
FCCP and CCCP are uncoupling agents that "short circuit" mitochondria. These carbonyl cyanide phenylhydrazones are lipid-soluble weak acids. Extensive charge delocalisation allows both the free acid and the ionised form to dissolve in non-polar phospholipids, thereby conducting protons and electrical charges across the mitochondrial inner membrane.
Atractyloside blocks the adenine nucleotide transporter in the inner membrane.
Ascorbate/TMPD together, although not inhibitors of the electron transport chain, have been used to elucidate the process of oxidative phosphorylation because they are able to feed electrons to Complex IV if the electron chain is blocked at an earlier stage. TMPD is tetramethyl phenylene diamine, an artificial electron carrier which is readily reduced by ascorbate, and oxidised by cytochrome c.
To help you understand how these inhibitors affect the different processes occurring in respiration, use the information provided above to fill in Table 2 below.
Table 2. Effect of inhibitors on the electron transport chain and ATP synthesis
Type of inhibitor | Electron movement down electron transport chain | O2 consumption | H+ movement into intermembrane space | pH gradient across membrane | ATP synthesis |
Inhibitors of components in electron transport chain |
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ATPase inhibitors
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Uncouplers |
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ATP transport inhibitors |
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Oxygen consumption by mitochondria can be measured quantitatively using an oxygen electrode. The following graph (Figure 2) illustrates how the order of electron transport chain components could be deduced using a variety of inhibitors. Check that you understand why oxygen uptake vaies in the way shown by Figure 2, by answering the following questions:
Figure 2
7. Why is a source of NADH required?
8. Why do the following compounds affect O 2 uptake?
a. FCCP
b. Rotenone
c. Succinate
d. Antimycin
e. Ascorbate +TMPD
The ratio of ATP synthesis to oxygen reduced (P/O ratio) can be calculated by adding a known amount of ADP to the reaction chamber and measuring the reduction in oxygen concentration
The following graphs (Figures 3 & 4) show results obtained with an oxygen electrode during two separate experiments with isolated mitochondria, which were incubated with buffered isotonic medium in the presence of various substrates and inhibitors. Explain what has happened in each situation. Why is there a small rate of oxygen consumption before ADP is added?
Figure 3 A P/O ratio of 2.5 was calculated for this experiment
Figure 4 A P/O ratio of 1.5 was calculated for this experiment
A young woman had a history of progressive muscle weakness since childhood. This weakness was especially noticeable on activities such as climbing stairs, running short distances, carrying heavy shopping bags, etc. She complained that drinking even small quantities of alcohol made her ill.
The patient was subjected to an exercise test; muscle fatigue rapidly ensued, with blood pyruvate and lactate rising to abnormally high levels. A muscle biopsy was examined. Some areas showed accumulation of neutral lipid in fibres that stained positively for succinate dehydrogenase. Muscle mitochondria prepared from the biopsy sample were studied in an oxygen electrode system and gave the following results:
Figure 5 | Figure 6 |
"Death on the Farm" in Biochemistry for the Medical Sciences: An Integrated Case Approach by S.J. Higgins, A.J. Turner, and E.J. Wood is related to this case and may be of help.