Assessing respiratory controlin metabolically active mitochondria by measuring changes in O2consumption rates in the of various substrates and respiratory poisons has providesinformation on how inhibitors affect the respiration rate. Isolated mitochondria are a good model for the study ofrespiratory control when attempting to understand the mechanisms of electrontransport and oxidative phosphorylation because results were produced whichallowed the quantitative analysis of respiration rate. For example, oligomycin was the greatestrespiratory inhibitor, reducing respiration rate from 0.
31 mmol/sec/ml when succinate is thesubstrate to 0.05 mmol/sec/ml. On theother hand, FCCP acts as an uncoupling agent increasing respiration rate to5.11 mmol/sec/ml.
However,this was while oligomycin was still in the system, providing a slightlyunexpected result. IntroductionMitochondria evolved around two billion yearsago when a precursor of the modern eukaryotic cell engulfed an a-proteobacterium (Lane and Martin, 2010). Mitochondriafulfil biosynthesis of important molecules such as lipids, amino acids, hemeand iron-sulfur clusters, and produce reactive oxygen species (Fontanesi, 2001), but their primaryfunction is to produce ATP through the citric acid cycle and oxidative phosphorylation.State IV respiration involves oxygen consumption in the presence of a substratesuch as succinate or glutamate in the absence of ADP or any respiratory poisons(Chance and Williams, 1955). StateIII respiration is ADP-stimulated and involves the catalysis of ADP and aninorganic phosphate using ATP synthase (Chance and Williams, 1955).
This permits protons to enter the matrix through openchannels from outside the inner membrane. The energy released from protonmovement is used to produce ATP. When ADP is exhausted, the system returns tostate IV, which is usually faster than the original rate because some ATP is brokendown by ATPase. The resulting ADP is re-phosphorylated (Chance and Williams, 1955). Researchershave supported this theoretical framework through use of animal tissue; for example,rat liver tissue (Graham, 2001). Animaltissue is a beneficial model respiratory control system for numerous reasons.First, animal tissue is more readily homogenised than plant tissue as plantcells contain cell walls that can be difficult to degrade (Hogeboom, 1955). Furthermore, separating organelles from highlystructured tissue such as muscle can be difficult because a high proportion ofthe mitochondria will remain trapped inside the tissue fragments.
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This suggest thatrat liver was an appropriate tissue to use in the isolation mitochondria in ourexperiment. Materials and MethodsPart I: Measuring the Phosphorus:Oxygen Ratioof State III Respiration using Succinate and Glutamate as SubstratesRespiration buffer (125 mM KCl, 1mM inorganicphosphate, 0.1 mM EGTA, 10 mM Tris Buffer, pH 7.4) and mitochondria were addedto the Rank Brothers Biological O2 Electrode Clark Cell andsuspended ensuring air was not introduced to the system. 10 mmol succinate produced state IVrespiration and 0.
6 mmol ADP producedstate III respiration, constructing a time versus oxygen graph on the DATAQdata-logger. This was repeated with 10 mmol glutamate to produce state IV respiration. The P:O ratios ofsuccinate and glutamate were calculated by measuring the decrease in oxygenconcentration during rapid bursts of state III respiration after adding theADP. Change in concentration was multiplied by the chamber volume (2.8 ml), sothat the answer (given in atoms of oxygen) can be related to the quantity ofADP added.
The quantity of oxygen in the chamber is calculated from publishedsolubility data at 25°C (0.237 mmol/ml). The P:O ratios for succinateand glutamate were calculated. The calculation for succinate P:O is shown inBox 1. Part II: Measuring the Effect of Reagents onRespiration Rate of Isolated Mitochondria A series of experiments were carried out withdifferent reagents in the Electrode Clark Cell containtaining respirationbuffer, mitochondria, succinate or glutamate to produce state IV respirationand ADP to produce state III respiration. The reagents used were 5 mmol malonate, 3×10-4 mmol antimycin, 1×10-6 mmol oligomycin, 5 mmol ascorbate, 0.6 mmol TMPD, 1×10-5 mmol rotenone and 0.
01 mmol FCCP. Then, respiration rate wascalculated by dividing the final minus initial oxygen concentration (%) afterADP was added by the final minus initial relative time point (seconds). Thefinal relative time point was determined by the point when ADP was exhaustedand state IV respiration set in. This gave the gradient of the change in oxygenconcentration. Results The results of the experiments are traced onWindaq software with voltage in the Clark cell proportional to theconcentration of dissolved O2 in the Clark cell.
Figure 1 shows thestate III respiration rate when succinate, glutamate, antimycin and ascorbateand TMPD were added to the system. A higher state III respiration rate isobserved when succinate is used to produce state IV respiration as compared toglutamate. The addition of antimycin, ascorbate and TMPD as reagentsdramatically increases the respiration rate to 0.95 mmol/sec/ml of mitochondria compared tono reagents.
Figure 2 shows one bar indicating a smallstate III respiration rate when rotenone is used and glutamate produces stateIV respiration. This indicates electron transport is somewhat inhibited as thevalues are much less than those for glutamate in Figure 1. The second bar showsthe effect when succinate is introduced to the system to restart state IVrespiration. This shows an increase in respiration rate when compared toglutamate and rotenone alone. Figure 3 shows the effect that FCCP andoligomycin have on state III respiration when succinate is used to producestate IV respiration. From the Figure, FCCP increases the respiration rate ascompared to succinate alone, whereas oligomycin decreases the rate ofrespiration to 0.
05 mmol/sec/ml ofmitochondria. The addition of FCCP to succinate and oligomycin dramatically increasesthe rate of respiration, well beyond the 0.31 mmol/sec/ml of mitochondria just succinate produces. Figure 4 shows malonate decreases the rate ofrespiration with increasing addition to the system. Glutamate allows therespiration rate to increase, which then decreases again with antimycin.Finally, ascorbate and TMPD allows the respiration rate to increase again, to agreater extent than either succinate or glutamate, without the addition ofreagents.
DiscussionFirst, from the results, it can be determinedthat rotenone, oligomycin, malonate, antimycin were respiratory poisons becausea decrease in respiration rate is observed when these reagents were added tothe system (Figures 2-4) compared to without reagents (Figure 1). Cyanide isanother known respiratory poison (Beasley and Glass, 1998) but this was notassessed in our experiment due to safety reasons. Electron transport inhibitorsprevent the passing of electrons from one complex to the other by physicallybinding to the electron transport chain. Rotenone inhibits NADH-linkedsubstrate oxidation in mitochondrial preparations of animal cells by binding tospecific sites responsible for inhibition between NADH dehydrogenase andcoenzyme Q (Singer, 1979). This is shown in Figure 2 by a respiration rate of 0.08 mmol/sec/ml of mitochondria. Quasteland Wooldridge (1928) first described the competitive inhibitory action ofmalonate on succinic dehydrogenase, explaining the reduced respiration ratewhen added to the mitochondria in our experiment (Figure 4).
When more malonateis added to the system, the respiration rate decreases further. This suggeststhat the active sites of succinic dehydrogenase became fully saturated withincreased malonate concentration, so succinate could not be targeted forreaction. Moreover, antimycin binds to the bc1 complex to preventthe reoxidation of cytochrome b by ubiquinone, therefore inhibiting electrontransport to cytochrome c (Esposti and Lenaz, 1982).
Second,FCCP is an uncoupling agent that causes a rapid increase in respiration ratebecause it increases the ionic permeability of phospholipid bilayers, inhibitingthe coupling between the electron transport chain and phosphorylationreactions, thus inhibiting ATP synthesis (Terada,H, 1990). FCCP enters the mitochondria in its protonated form, dischargingthe pH gradient and then leaves as a negatively charged molecule (Terada, H, 1990). This destroys themembrane potential and catalyses the movement of huge numbers of protons. This isseen in one of the bars in Figure 3, where the respiration rate increases to5.11 mmol/sec/ml. However,the succinate and FCCP bar doesn’t show the expectedresults, showing the experiment has some limitations. This could be due toexperimental errors whereby the chamber was not washed effectively betweenexperiments, meaning remnants of respiratory poisons were still present.
Finally, oligomycin is a phosphorylation inhibitor of F0F1ATPaseby binding to a 23 kd polypeptide in the F0 baseplate in the enzyme, preventingprotons passing back into the mitochondria and inhibiting proton pumps asgradients exceed their operational threshold (Acidemia and Branched-Chain, 2010). This is shown in Figure 3where the respiration rate with oligomycin is minimal. A furtherweakness of our experiment is gender of the rat was not taken into account.
Anexperiment carried out by Caligioni, (2009) describes that using male mice is favouredbecause the oestrous cycle in females can release hormones that causecomplications in experiments. This is something to take into account in futureexperiments. In conclusion, this experiment was a success as the aims wereeffectively met.
Changes in oxygen consumption were measured in the presence orabsence of substrates and respiratory poisons. From the results, oligomycin wasthe greatest respiratory inhibitor while FCCP massively increased respirationrate. It can be determined that isolatedmitochondria are a good model in the study of respiratory control because theresults showed that respiration rate changes when mitochondria are in thepresence of different reagents.
