Reference no: EM132301088

PROGRAM: ANALYSING ISOLATED ORGANELLES

INVESTIGATING THE FUNCTIONOF ISOLATED MITOCHONDRIA

INTRODUCTION

The living cell is a dynamic and highly organized structure many aspects of which can be investigated in the intact cell using microscopy. However, if we wish to investigate the function or properties of specific organelles or macromolecules in detail, they must first be separated from other cellular components.

In this activity you will investigate the oxygen consumption of isolated mitochondria. By adding particular reagents to mitochondrial suspensions and monitoring oxygen consumption, information can be inferred about the function and organization of the respiratory complexes and oxidative phosphophorylation.

Mitochondria! diseases are associated with defects in respiration and oxidative phosphorylation. The basis of these defects can be investigated in isolated mitochondria from patients using the approaches illustrated in this program.

OBJECTIVES
At the end of this activity you will be able to assess mitochondrial respiratory function using polarography and underdstand the relationship between electron transport, oxygen consumption,

ATP production and the chemiosmotic theory.

ACTIVITY OVERVIEW
1. Complete pre-prac quiz
2. Calibrate the oxygen electrode
3. Measure mitochondrial respiration using malate/pyruyate
4. Calculate the parameters (a) respiration rate (b) respiratory control ratio and (c) P:0 ratio for a mitochondrial preparation.

PART 1 MEASUREMENT OF OXYGEN CONSUMPTION BY ISOLATED

MITOCHONDRIA

Investigate the oxygen consumption (respiration) rates of mitochondria using the oxygen electrode. You will be provided with isolated beef heart mitochondna prepared as described above.

You should now be familiar with the components, assembly and use of an oxygen electrode from watching the video clips in the OxPhos program. Assemble your oxygen electrode following the instructions in the manual and by watching a video on the unit website. It may be necessary at some stage in the laboratory for you to disassemble and reassemble the electrode should the membrane need replacing. The membrane can become clogged and leading to a sluggish response changes ion oxygen levels.

You will need to calibrate the oxygen electrode before you can produce meaningful data. Once the electrode is calibrated you will monitor the oxygen consumption of samples of the mitochondria after addition of reagents in a sequential manner. After acquiring and saving your data you will perform calculations to determine (1) rates of respiration; (2) respiratory control ratio; and (c) P-.0 ratio, and complete Results Table 1.

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-malete or succmate as substrates.

3. Comment on the RCR for your preparation of mitochondria. using pyruvale-malate or suer:mate 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:0 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 expect there to be a difference between pyruvate-malate and succinate.

DAY 3 - TESTING THE EFFECTS OF INHIBITORS ON RESPIRATION
Data interpretation: use of dear 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 mitochondnal icaj.ei
• Appreciate the use of CNE to examine mrtochondnal protein comptexes

ACTIVITY OVERVIEW
1. Pre-prac Quiz
2. Assemble and calibrate the oxygen electrode
3. Measure oxygen consumption rates of thawed mitochondna
4. Monitor the effects of inhibitors and uncoupiers on mibchondnal respiration
5. Complete Results Tables 2 and 3, calculating (a) rate of respeahon, (b) respiratory control rata

PART 1 - TESTING THE EFFECTS OF INHIBITORS AND UNCOUPLERS ON RESPIRATION

PART 2. USE OF NATIVE ELECTROPHORESIS TO EXAMINE MITOCHONDRIAL 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 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.

3. How would you set up experimental assays using intact mitochondria with various subst tes and inhibitors, to measure the activity of the following enzymes spectrophotornetricalty? rates [Note: that the inter-conversions of NADH/NAD4 and cyt erdeyt cmd can each spectrophotometrically measured
(a) Complex I
(b) Complexes II and III together
(c) Complex IV

Attachment:- lab.rar