Assessing citric acid cycle and oxidative phosphorylation. State

Assessing respiratory control
in metabolically active mitochondria by measuring changes in O2
consumption rates in the of various substrates and respiratory poisons has provides
information on how inhibitors affect the respiration rate. Isolated mitochondria are a good model for the study of
respiratory control when attempting to understand the mechanisms of electron
transport and oxidative phosphorylation because results were produced which
allowed the quantitative analysis of respiration rate. For example, oligomycin was the greatest
respiratory inhibitor, reducing respiration rate from 0.31 mmol/sec/ml when succinate is the
substrate to 0.05 mmol/sec/ml. On the
other hand, FCCP acts as an uncoupling agent increasing respiration rate to
5.11 mmol/sec/ml. However,
this was while oligomycin was still in the system, providing a slightly
unexpected result.

 

Introduction

Mitochondria evolved around two billion years
ago when a precursor of the modern eukaryotic cell engulfed an a-proteobacterium (Lane and Martin, 2010). Mitochondria
fulfil biosynthesis of important molecules such as lipids, amino acids, heme
and iron-sulfur clusters, and produce reactive oxygen species (Fontanesi, 2001), but their primary
function is to produce ATP through the citric acid cycle and oxidative phosphorylation.
State IV respiration involves oxygen consumption in the presence of a substrate
such as succinate or glutamate in the absence of ADP or any respiratory poisons
(Chance and Williams, 1955).  State
III respiration is ADP-stimulated and involves the catalysis of ADP and an
inorganic phosphate using ATP synthase (Chance and Williams, 1955). This permits protons to enter the matrix through open
channels from outside the inner membrane. The energy released from proton
movement is used to produce ATP. When ADP is exhausted, the system returns to
state IV, which is usually faster than the original rate because some ATP is broken
down by ATPase. The resulting ADP is re-phosphorylated (Chance and Williams, 1955).

 

Researchers
have supported this theoretical framework through use of animal tissue; for example,
rat liver tissue (Graham, 2001). Animal
tissue is a beneficial model respiratory control system for numerous reasons.
First, animal tissue is more readily homogenised than plant tissue as plant
cells contain cell walls that can be difficult to degrade (Hogeboom, 1955). Furthermore, separating organelles from highly
structured tissue such as muscle can be difficult because a high proportion of
the mitochondria will remain trapped inside the tissue fragments. This suggest that
rat liver was an appropriate tissue to use in the isolation mitochondria in our
experiment.

 

Materials and Methods

Part I: Measuring the Phosphorus:Oxygen Ratio
of State III Respiration using Succinate and Glutamate as Substrates

Respiration buffer (125 mM KCl, 1mM inorganic
phosphate, 0.1 mM EGTA, 10 mM Tris Buffer, pH 7.4) and mitochondria were added
to the Rank Brothers Biological O2 Electrode Clark Cell and
suspended ensuring air was not introduced to the system. 10 mmol succinate produced state IV
respiration and 0.6 mmol ADP produced
state III respiration, constructing a time versus oxygen graph on the DATAQ
data-logger. This was repeated with 10 mmol glutamate to produce state IV respiration. The P:O ratios of
succinate and glutamate were calculated by measuring the decrease in oxygen
concentration during rapid bursts of state III respiration after adding the
ADP. Change in concentration was multiplied by the chamber volume (2.8 ml), so
that the answer (given in atoms of oxygen) can be related to the quantity of
ADP added. The quantity of oxygen in the chamber is calculated from published
solubility data at 25°C (0.237 mmol/ml). The P:O ratios for succinate
and glutamate were calculated. The calculation for succinate P:O is shown in
Box 1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Part II: Measuring the Effect of Reagents on
Respiration Rate of Isolated Mitochondria

A series of experiments were carried out with
different reagents in the Electrode Clark Cell containtaining respiration
buffer, mitochondria, succinate or glutamate to produce state IV respiration
and 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 was
calculated by dividing the final minus initial oxygen concentration (%) after
ADP was added by the final minus initial relative time point (seconds). The
final relative time point was determined by the point when ADP was exhausted
and state IV respiration set in. This gave the gradient of the change in oxygen
concentration.

 

 

 

 

 

 

 

 

 

Results

The results of the experiments are traced on
Windaq software with voltage in the Clark cell proportional to the
concentration of dissolved O2 in the Clark cell. Figure 1 shows the
state III respiration rate when succinate, glutamate, antimycin and ascorbate
and TMPD were added to the system. A higher state III respiration rate is
observed when succinate is used to produce state IV respiration as compared to
glutamate. The addition of antimycin, ascorbate and TMPD as reagents
dramatically increases the respiration rate to 0.95 mmol/sec/ml of mitochondria compared to
no reagents.

 

 

Figure 2 shows one bar indicating a small
state III respiration rate when rotenone is used and glutamate produces state
IV respiration. This indicates electron transport is somewhat inhibited as the
values are much less than those for glutamate in Figure 1. The second bar shows
the effect when succinate is introduced to the system to restart state IV
respiration. This shows an increase in respiration rate when compared to
glutamate and rotenone alone.

 

Figure 3 shows the effect that FCCP and
oligomycin have on state III respiration when succinate is used to produce
state IV respiration. From the Figure, FCCP increases the respiration rate as
compared to succinate alone, whereas oligomycin decreases the rate of
respiration to 0.05 mmol/sec/ml of
mitochondria. The addition of FCCP to succinate and oligomycin dramatically increases
the rate of respiration, well beyond the 0.31 mmol/sec/ml of mitochondria just succinate produces.

 

 

Figure 4 shows malonate decreases the rate of
respiration with increasing addition to the system. Glutamate allows the
respiration rate to increase, which then decreases again with antimycin.
Finally, ascorbate and TMPD allows the respiration rate to increase again, to a
greater extent than either succinate or glutamate, without the addition of
reagents.

 

 

Discussion

First, from the results, it can be determined
that rotenone, oligomycin, malonate, antimycin were respiratory poisons because
a decrease in respiration rate is observed when these reagents were added to
the system (Figures 2-4) compared to without reagents (Figure 1). Cyanide is
another known respiratory poison (Beasley and Glass, 1998) but this was not
assessed in our experiment due to safety reasons. Electron transport inhibitors
prevent the passing of electrons from one complex to the other by physically
binding to the electron transport chain. Rotenone inhibits NADH-linked
substrate oxidation in mitochondrial preparations of animal cells by binding to
specific sites responsible for inhibition between NADH dehydrogenase and
coenzyme Q (Singer, 1979). This is shown in Figure 2 by a respiration rate of 0.08 mmol/sec/ml of mitochondria. Quastel
and Wooldridge (1928) first described the competitive inhibitory action of
malonate on succinic dehydrogenase, explaining the reduced respiration rate
when added to the mitochondria in our experiment (Figure 4). When more malonate
is added to the system, the respiration rate decreases further. This suggests
that the active sites of succinic dehydrogenase became fully saturated with
increased malonate concentration, so succinate could not be targeted for
reaction. Moreover, antimycin binds to the bc1 complex to prevent
the reoxidation of cytochrome b by ubiquinone, therefore inhibiting electron
transport to cytochrome c (Esposti and Lenaz, 1982).

 

Second,
FCCP is an uncoupling agent that causes a rapid increase in respiration rate
because it increases the ionic permeability of phospholipid bilayers, inhibiting
the coupling between the electron transport chain and phosphorylation
reactions, thus inhibiting ATP synthesis (Terada,
H, 1990). FCCP enters the mitochondria in its protonated form, discharging
the pH gradient and then leaves as a negatively charged molecule (Terada, H, 1990). This destroys the
membrane potential and catalyses the movement of huge numbers of protons. This is
seen in one of the bars in Figure 3, where the respiration rate increases to
5.11 mmol/sec/ml. However,
the succinate and FCCP bar doesn’t show the expected
results, showing the experiment has some limitations. This could be due to
experimental errors whereby the chamber was not washed effectively between
experiments, meaning remnants of respiratory poisons were still present.
Finally, oligomycin is a phosphorylation inhibitor of F0F1ATPase
by binding to a 23 kd polypeptide in the F0 baseplate in the enzyme, preventing
protons passing back into the mitochondria and inhibiting proton pumps as
gradients exceed their operational threshold (Acidemia and Branched-Chain, 2010). This is shown in Figure 3
where the respiration rate with oligomycin is minimal.

 

A further
weakness of our experiment is gender of the rat was not taken into account. An
experiment carried out by Caligioni, (2009) describes that using male mice is favoured
because the oestrous cycle in females can release hormones that cause
complications in experiments. This is something to take into account in future
experiments.

 

In conclusion, this experiment was a success as the aims were
effectively met. Changes in oxygen consumption were measured in the presence or
absence of substrates and respiratory poisons. From the results, oligomycin was
the greatest respiratory inhibitor while FCCP massively increased respiration
rate. It can be determined that isolated
mitochondria are a good model in the study of respiratory control because the
results showed that respiration rate changes when mitochondria are in the
presence of different reagents.