STUDIES ON COUPLING OF PROTON MOTIVE FORCE PROMOTED MITOCHONDRIAL ELECTRON TRANSPORT TO ATP SYNTHESIS

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Diana Pearson
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1 STUDIES ON COUPLING OF PROTON MOTIVE FORCE PROMOTED MITOCHONDRIAL ELECTRON TRANSPORT TO ATP SYNTHESIS According to the chemiosmotic theory, oxidation of respiratory substrates, phosphorylation of ADP to ATP as well as ion transport into and from mitochondria are strongly coupled via a proton concentration gradient which builds up between the two sides of the inner mitochondrial membrane in the course of the electron transport mediated by the mitochondrial respiratory chain complexes. Gibbs' free energy generated by the redox reactions of the mitochondrial respiratory chain provides the driving force to transport H + from the matrix ( mx ) to the intermembrane space ( im ) of mitochondria and build up the above mentioned concentration gradient of the H + ions and also an electrical potential difference ( E m ) which is generated directly due to the charge separation between the two faces of the inner membrane. The sum of the two energies is called the proton motive force (proton electrochemical potential): 2.3 RT H + im P= E m + lg = E m - 0,06(pH im - ph mx )= E m +0,06 ph F H + mx Phosphorylation of ADP by inorganic phosphate is facilitated by the proton motive force. This hence constitutes the coupling of the oxidative phosphorylation to the terminal oxidation. Compounds that hamper the formation or the maintenance of the above mentioned proton gradient abolish the driving force for ATP synthesis and selected active transport mechanisms. This also abrogates the coupling between reactions of energy production and usage. These compounds are referred to as uncouplers and they are usually of lipophilic character with optimal proton binding capacity near ph 6.5 (weak acids; e.g. 2,4-dinitrophenol). OXIDATION OF SUBSTRATES AND ATP GENERATION 1. Respiratory control and the P/O ratio Under the condition of intact mitochondria oxidizing respiratory substrates (in the course of phosphorylating ADP to ATP), the rate of oxygen consumption and the quantity of the ATP already synthesized can be both assayed in parallel. The rate of substrate oxidation measured under optimal conditions (respiratory substrate at saturation concentration, inorganic phosphate and oxygen dissolved in a buffered isotonic medium at neutral ph) depends only on whether ADP is present or not. The accelerating effect on substrate oxidation 1

2 of ADP is referred to as the acceptor control, or respiratory control: in the absence of ADP the rate of respiration is low (resting respiration), but it becomes higher when ADP, even at low concentration, is given. After ADP gets phosphorylated, the oxygen uptake returns to a low rate. The proportion of the rate of oxygen uptake measured in the presence of ADP to the rate measured after ADP is consumed is characteristic of the efficiency of the coupling that exists between the mitochondrial respiration and ATP synthesis, and is called the respiratory control ratio (RCR): The rate of oxygen uptake in the presence of ADP RCR = 1 The rate of oxygen uptake in the absence of ADP In case any uncoupler is present, or the inner membrane is so-called leaky, both the RCR and the P/O decrease, thus ADP fails to accelerate respiration, and no net ATP gets synthesized, although the rate of oxygen uptake is still high. When in well-coupled mitochondria the ATP synthesis is inhibited by the antibiotic oligomycin, respiration also becomes suppressed since the proton motive force generated by respiration reaches a limit represented by the Gibbs' free energy change of substrate oxidation (and also because the matrix becomes alkaline); a similar effect is observed in the absence of phosphate or ADP. Transport of ADP from the cytoplasm to the matrix via the ADP:ATP translocase of the inner mitochondrial membrane can be inhibited by atractylosides; these compounds also inhibit the respiration, although the proton motive force remains still high. Respiration can then be re-established by an uncoupler or upon launching an ion transport (e.g. uptaking Ca 2+ ). 2. Inhibiting the respiratory chain Substances, known as site-specific inhibitors of the respiration, lower the oxygen uptake and they are applied when studying the characteristics of the sites oxidizing specific substrates. Due to the lack of energy production, neither the generation of a proton motive force, nor the synthesis of ATP (nor an active ion transport) can be expected, and respiration will not be re-established either, even when ADP or an uncoupler is given. In the presence of a specific inhibitor of the electron transport chain, all the components of the chain which are localized between the binding sites of the substrate and the specific inhibitor become reduced, while all the further components towards the oxygen binding site become oxidized. The most important respiratory inhibitors are: CN -, CO, N 3 - (inhibiting cytochrome oxidase), antimycin-a (acts between cytochrome b and c 1 ), malonate (inhibiting succinate dehydrogenase) and rotenone (inhibiting NADH dehydrogenase). 2

3 3. Principle of the polarographic assay of dissolved oxygen Oxygen consumption during respiration is recorded by using a Clark-type oxygen electrode composed of a reference electrode (silver/silver chloride) and a platinum electrode (negative relative to the reference electrode). Both are immersed in a highly concentrated (4 M?) KCl solution. The apparatus is separated from the reaction vessel by a polyethylene membrane, which permits dissolved oxygen entering the electrode surface layer from the reaction mixture, and here, if a voltage is imposed across the two electrodes, oxygen undergoes an electrolytic reduction. When current is plotted as a function of polarizing voltage, first an increase in current (at about 0.4 V), then a plateau region (between 0.5 and 0.8 V) can be observed. The increase in current is caused by the increasing number of oxygen molecules participating in the transport of charges. Then, if voltage is further elevated, the number of oxygen molecules involved in transport reaches a limit determined by their rate of diffusion, which is proportional to the concentration gradient of oxygen established between the bulk of solution and electrode surface ('diffusion-limited current' in the plateau region). By this means, at 0.6 V constant polarization voltage current becomes directly proportional to the concentration of oxygen. Current recorded under these conditions is used to calculate changes in oxygen concentration during respiration. The reaction vessel is kept isothermic at a constant temperature and the reaction mixture is stirred continuously during the reaction by the use of a plastic-encased flea driven by a magnetic stirrer to facilitate establishment of the equilibrium between the concentrations of oxygen in the reaction mixture and the electrode space. Reactions are carried out in a closed vessel, so no air bubbles are allowed to be trapped above the medium, and on the inner wall of the vessel. Reactants are carefully pipetted to a hole on the stopper of the vessel, and the force of stirring will drive them inside. After each experiment the vessel is emptied by suction aspiration of the fluid and washed by repeated water rinsing. Electronic equipment composed of the polarizing voltage source and sensitivity control adjustments are arranged as shown in Fig. 1. Oxygen current is converted to voltage change and recorded by a potentiometric recorder at its highest sensitivity (2 mv/full scale). 3

4 Fig.1. Scheme of the polarizing voltage and sensitivity control of an oxygen electrode. The variable resistor R 1 is the sensitivity control, R 2 supplies a polarizing voltage of 0.6 V, R 3 is for zero offset control. REAGENTS, PREPARATIONS AND EQUIPMENT Preparation of mitochondria (needed directly in advance of the laboratory lesson): Sacrifice a rat by cervical dislocation, excise the liver, rinse it and then place it into the icecold Isolating Buffer. Chop it into small pieces, rinse it again, then add a 4-fold volume of Isolating Buffer, and then homogenize it carefully in a Potter-Elvehjem homogenizer (spherical pestle, large clearance), with a few strokes. Repeat homogenization using a tighter pestle of cylindric shape, with a small clearance. Centrifuge homogenate at 600 x g at 0 o C for 8 min, then decant the supernatant into a clean centrifuge tube (the sediment contains largely nuclei and cell debris and it is discarded). Centrifuge supernatant at x g for 20 min to pellet mitochondria. Store mitochondria in the form of pellet until use. When needed, suspend the mitochondria carefully in ice-cold Isolating Buffer to about mg prot./ml, and keep it on ice. Sample: Mitochondrial suspension at a protein concentration of mg/ml. Reagents: Incubating Buffer (called 'Medium'): 80 mm KCl, 20 mm Tris, 1 mm EGTA, 10 mm KH 2 PO 4, ph 7.2. Reagents glutamate (ph= 7.2) malate (ph= 7.2) Concentration of stock solution Volume added Final concentration 0.7 M 20 µl 4.67 mm 0.3 M 20 µl 2 mm 4

5 succinate 0.75 M 20 µl 5 mm (ph= 7.2) ADP (ph= 7.2) 50 mm 20 µl 0.33 mm DNP 10 mm 20 µl mm oligomycin (dissolved in ethanol) 0.08 mg/ml (M=805 g/mol) 9.9*10-11 M = 9.9*10-8 mm= 9.9* 10-5 µm=0.099 pmol malonate 100 mm 20 µl mm (ph= 7.2) KCN 100 mm 20 µl mm atractyloside 5 mg/ml (M= 803 g/mol) 20 µl 6.2 * 10-6 M= 6.2 * 10 - mm= 6.2 µm Na-dithionite (solid) Equipment Reaction vessel with a stopper, a teflon-coated stirring flea and a magnetic stirrer, an attached oxygen electrode with a polarizer unit (needs a 1.5 V battery and the polarizer output adjusted to 0.6 V) and a potentiometric recorder. EXPERIMENTAL PROCEDURE WARNING! You are working with highly toxic substances. Be very careful! Use separate tips for each reagent to avoid the contamination of the stock solutions. Indicate in your recordings the exact time a reagent was given at. Avoid bubbles remaining in, or entering the vessel after you start the experiment. Calibration of the apparatus The initial saturation concentration of oxygen in the medium as a function of temperature is shown in Fig. 2. To use this plot for computing the actual oxygen concentrations, one needs to know the exact temperature of the medium at any given point. During the calibration procedure a calibration curve on the potentiometer is recorded between the limits of the saturation concentration of oxygen and no oxygen present at all (achieved by the addition of dithionite). To launch the calibration procedure, switch the polarizing unit as well as the potentiometric recorder on and pipette 3.5 ml incubation medium into the reaction vessel. Place the stopper (lid) on the vessel to isolate the medium from air, then switch the magnetic stirrer on (at the lowest speed), start the recorder and the pen (at the chart speed of 60 cm/h 5

6 (=1 cm/min), at 2 mv/full scale). When the pen is stabilized add a few grains of sodium dithionite close to the hole on the stopper, turn the stopper slightly to permit dithionite entering the vessel. The pen is now moving towards 0. When it is stabilized, adjust it to 0 unit (use the zero control knob on the recorder). Zero oxygen concentration is now set. To adjust the recorder pen to saturating oxygen concentration, first remove the stopper, and then carefully clean up the vessel and stopper by repeated rinsing with deionized water (use water aspiration, but be careful with the electrode membrane located on the inner wall of the vessel at the side arm). Pipette a fresh batch of medium into the vessel, close the stopper (start the stirrer and the recorder), and when the pen is stabilized, adjust its position to 100 units (use the sensitivity knob on the polarizer unit). After this calibration procedure is completed, saturation oxygen concentration will be set to 100 units on the recorder. Exp. #1: Determination of the respiratory control and the ADP/O (P/O) ratio The experiment may be carried out using the substrate succinate, or glutamate plus malate. Proposed procedure: Add 3 ml medium Add 20 µl substrate (glutamate plus malate or succinate) close the vessel, set the pen at position 100, and wait until no more change occurs in pen position (no oxygen consumption); Add 50 µl mitochondria and record the slope of the line on the recorder; Add 20 µl ADP: Record the oxygen uptake rates. After ADP is utilized and respiration is declined, give ADP again, and record respiration one more time. Calculate the P/O ratio after adding ADP each occasion (see attached sheet); Add a few grains of dithionite to demonstrate that oxygen is still present in the medium. Exp. #2: Effects of ADP, atractyloside, DNP or KCN on respiration The experiment may be carried out using the substrate succinate, or glutamate plus malate. Proposed procedure: Add medium (3.0 ml) + substrate (20 l), close the vessel with the stopper, and wait until no more change occurs in pen position (roughly 1 min); Add 50 l mitochondria piercing through the stopper and after respiration is stabilized; Add 20 l ADP and record the oxygen uptake rates; Add 20 l atractyloside to inhibit the adenine-nucleotide translocase; Add 20 l DNP to uncouple respiration from phosphorylation, check whether DNP indeed restored the fast oxygen consumption; 6

7 Add 20 l KCN to inhibit complex IV and hence the mitochondrial respiration; Add a few grains of dithionite to demonstrate that oxygen is still present in the medium. Exp. #3: Inhibition of succinate oxidation by malonate Proposed procedure: Add medium (3.0 ml) + succinate (20 l), close the vessel with stopper, and wait until no more change occurs in pen position (roughly 1 min); Add 50 l mitochondria piercing through the stopper and after respiration is stabilized; Add 20 l ADP after recording the accelerated respiration; Add 20 l malonate to competitively inhibit the succinate dehydrogenase; Add 20 l succinate to re-establish the mitochondrial respiration; Add a few grains of dithionite to demonstrate that oxygen is still present in the medium. Exp. #4: Effects of ADP, oligomycin, DNP and KCN on respiration Proposed procedure: Add medium (3.0 ml) + glutamate plus malate (20 l), close the vessel with stopper, and wait until no more change occurs in pen position (roughly 1 min); Add 50 l mitochondria piercing through the stopper and when respiration is stabilized; Add 20 l ADP after recording the accelerated respiration; Add 20 l oligomycin to inhibit the ATP-synthase and block the mitochondrial respiration; Add 20 l DNP to re-establish the mitochondrial respiration; Add a few grains of dithionite to demonstrate that oxygen is still present in the medium. SCOPES OF THIS LABORATORY LESSON The main objectives of this class are to study and demonstrate the mechanism of the mitochondrial respiration and evaluate the effects of different inhibitors on the mitochondrial respiration with an emphasis on the effect of oligomycin, an ATP synthase inhibitor. Additionally, a further aim is to determine the P/O ratio and RC in the presence of succinate or glutamate plus malate. 7

8 EVALUATION OF THE RESULTS 1. Calculate the rates of 'resting' and 'active' respiration in oxygen/mg prot./min for the respiratory substrates used. 2. Calculate the RC values for the two substrates. 3. Calculate P/O (ADP/O) for the substrate used. 4. Evaluate the effects of the respiratory inhibitors and the uncoupler on the mitochondrial respiration, the ATP synthesis and the mitochondrial proton gradient! Fig.2. Oxygen content of water saturated with air as a function of temperature. 8

9 O 2 100% Fig.3. Determination of the ADP/O (P/O) ratio using the experimental results. CALCULATION OF THE ADP/O (P/O) RATIO (See Fig. 2. and 3. for details) Final volume: Temperature: Saturating conc. of O 2 at this temp.: Total oxygen content ('X'): Oxygen used for ADP ('Y'):...units = Substrate for the respiration: Concentration of the stock solution: Volume added: Concentration of the ADP stock solution: Volume added: Amount of ADP used: ml 0 C mole/ml mole mole mm l mm ml mole ADP, mole... ADP/O (P/O) ratio: = = 2* O 2, mole 2*... 9

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