Reference no: EM132325938
PROGRAM: ANALYSING ISOLATED ORGANELLES
INVESTIGATING THE FUNCTION OF ISOLATED MITOCHONDRIA
Read the background information on the purification of mitochondria from beef heart. VIDEO - 'Using the Clarke electrode.
OBJECTIVES
At the end of this activity you will be able to assess mitochondrial respiratory function using polarography and understand the relationship between electron transport, oxygen consumption, ATP production and the chemiosmotic theory.
You are now ready to perform your first experiment with Isolated mitochondria. All additions to the chamber through the plunger are made using the white flexible tips.
1. Frozen aliquots of isolated mitochondria have been transferred from an - 80°C storage freezer to the teaching laboratory on dry ice. When you are ready to use the mitochondria place a single snap cap tube containing frozen mitochondria into a floating test tube holder and incubate in the 30°C water bath until thawed (about 1 minute). Record the annotation on the cap. Once thawed transfer to wet ice. Using a 1 ml pipette (blute tip) gently pipette the suspension up and down several times with a blue tip. Store the aliquot at wet ice at all times. Why is this important?
2. In the Biograph menu bar select "Page' > '2' to open a new page to record data. In the 'Digital Mu'Omelet' window select, "20 min*. Biograph will record data for 20 mins and stop. Activate 'Markers enabled" to allow you to use the keyboard to record when addition are made to the experiment.
3. Using a 1 ml pipette and 2 ml of incubation buffer to the incubation chamber and assembly the plunger ensuring bobbles are expelled. Unfortunately the buffer readily forms bubbles due the presence of bovine serum albumin. Allow the votage to stabilize to -10 mv and select "GO" on the Digital MultiMeter panel.
Incubation contains 0.25 M sucrose, 50 mM KCL, 5mM Tris-HCL, 1mM EGTA, 5mM K-PO pH 7.4, 4mg/ml fatty acid free bovine serum albumin.
What is the rote of sucrose in the incubation buffer?
4. Monitor the trace for about 1 minute. If the signal appears relatively stable add 50 μl of the mitochondria suspension into the incubation chamber without introducing bubbles. Immediately select ‘M' on the keyboard to record when the mitochondria where added.
Observe the trace for -1 min to ensure a steady signal.
5. Add 100 μl of substrate (0.1 M pyruvate plus 0.1 M )into the incubation chamber and mark the addition using the 'S' key. Observe le trace for -1 minute of until a steady state is observed. (i.e. straight line).
Which respiratory state have the mitochondria entered?
6. Add 10 μl of 50mM ADP and Mark this addition using the 'A' * key. Observe the trace until a new steady state has been achieved.
Which respiratory slate have the mitochondria entered?
Eventually the steady state will change again
Which respiratory state have the mitochondria entered? What would happen to the trace if you added a further aliquot of ADP?
QUESTIONS
1. Comment on the reproducibility of the measured rates for each of the substrates used.
2. Comment on any differences in the mean rates of respiration comparing pyruvete-malate or succinate as substrates.
3. Comment on the RCR for your preparation of mitochondria, using pyruvate-malate or succinate as substrates. Do you consider yours to be a well coupled preparation?
4. Comment on the P:O ratio measured for each of pyruvate-malate and succinate as substrates. Are the P:O ratios of the anticipated order of magnitude? Are there systematic differences between the P:O ratios measured for the two types of substrates used? Explain why you would exped there to be a difference between pyruvate-malate and succinate.
TESTING THE EFFECTS OF INHIBITORS ON RESPIRATION
Data interpretation: use of clear native electrophoresis to examine mitochondria) protein complexes
OBJECTIVES
At the end of day 3 you should be able to:
• Explain the effects of inhibitors and uncouplers on mitochondrial respiration
• Appreciate the use of CNE to examine mitochondria protein complexes
QUESTIONS
1. Is it necessary that mitochondria are 'coupled' for experiments with respiratory Inhibitors? Expain your answer. Comment on the respiratory activity of mitochondria determined in week 2 compared to that obtained in week 3. If there are differences suggest reasons for the differences.
2. In your experiment you mentioned Oxygen consumption to investigate mitochondria function. Using an additional % assay It is possible to simultaneously measure the amount of ATP present on the chamber of the oxygen electrode. The following indentified plot shows the oxygen consumption by mitochondria after different addition. Complete the graph (below) le show how you think the relative ATP levels (on a scale of 0-1) might change during the course of the experiment CCCP is a protonophore.
3. In conjunction with the data above, complete the following diagram to show the sites at which substrates donate electrons to, and inhibitors act on, the electron transport chain of mitochondria.
Complex ||
↓
NADH Complex → CoQ → Complex III → cyt c → Complex IV → O2
4. How would you set up experimental assays, using intact Mitochondria with various substrates, and inhibitors, to measure the activity of the following enzymes spectrophotometrically?
[Note: that the inter-conversions of NADH/NAD. and cyt Cos/cyt Cred can each be measured spectrophotometrically]
(a) Complex I
(b) Complexes II and III together
(c) Complex IV
Attachment:- Mito inhibitors lab.rar