Exercise 8.7 - Respiration of Mitochondria

LEVEL III

Materials

Gilson (or Warburg) Respirometer at 37° C

• Mitochondria suspension from Exercise 8.4
  
• Krebs Phosphate Ringers (KPR)
  
• 10% (w/v) Glucose
  
• 0.39% (w/v) Sodium azide
  
• 18.4 mg% (w/v) Dinitrophenol (DNP)
  
• 6.64% (w/v) Sodium Malonate
  
• 10% (w/v) KOH

1. When you enter the lab, check that the reference chambers of the Gilson respirometer are filled with the appropriate solution (usually formaldehyde). Turn on the instrument and equilibrate the temperature.

2. Line up fourteen reaction vessels and ensure that each is clean, free of cracks and has all the necessary springs (4 each), stoppers and vent tubes handy.

3. Use a micropipette to carefully place 0.2 ml of 10% KOH in the center well of each. It is extremely important that no KOH spill into the main reaction vessel,as this will destroy the mitochondria placed in the vessel.

4. Place 2.0 ml of KPR in the main vessel of flasks #1 and #2.

5. Place 0.5 ml of KPR in the main vessel of flasks #3 and #4.

6. Place 0.2 ml of KPR in the main vessel of flasks #5 through #12.

7. Place 0.1 ml of KPR in the main vessel of flasks #13 and #14.

8. Place 1.0 ml of mitochondria suspension in the main vessels of flasks #3 through #14.

9. Add 0.5 ml of glucose to the side arm of flasks #3 through #12.

10. Finally, add the following to the main vessels of the indicated flasks:

    #5 and #6	0.3 ml of glucose
    #7 and #8	0.3 ml of sodium azide
    #9 and #10	0.3 ml of DNP
    #11 and #12	0.3 ml of sodium malonate

11. Cut fourteen 2 cm squares from filter paper and make small fans out of each. Place one piece each into the center well. Doing this will increase the surface area available for CO absorption by the KOH.

12. Carefully attach the sidearm stopper (or closed vent tube) to the flasks. Attach the flasks to the manometers and secure with springs. Lower the flasks into the water bath.

13. Allow all of the reaction flasks to temperature equilibrate for 5 minutes. During this time, set all of the micrometers to a reading of 500.

14. Add the contents of the sidearms to the main vessels by lifting the entire manometer assembly from the water bath and gently tipping the flask.

    BE CAREFUL NOT TO SPILL ANY CONTENTS INTO OR OUT OF THE CENTER WELL.

15. Place all of the flasks back into the water bath and close all valves of the respirometer. This is the zero point for your data - record the time.

16. Turn on the shaking motor and adjust the speed to obtain a gentle swirl of the flask contents.

17. After 10 minutes, adjust the manometer fluid to the original starting line by turning the appropriate micrometer. Record the micrometer readings for each of the fourteen flasks.

18. Repeat the micrometer readings at 10 minute intervals until a stable slope is obtained for the respirometers. This will generally be within 40-60 minutes from the zero point. Record all readings.

19. For each reading, subtract the original value of 500. Average the readings obtained in flasks #1 and #2 and subtract this average from the averages of #3/#4, #5/#6, #7/#8, #9/#10, #11/#12 and #13/#14.

20. Plot the change in gas volume (oxygen consumption) for each of the replicate conditions. 10

Tubes 1 and 2 are thermobarometers. Any changes in gas volume within these tubes reflects changes in the temperature and/or barometric pressure. Tubes 3/4 and 5/6 represent all of the conditions for oxidative phosphorylation (respiration). Tubes 7/8 are inhibited by azide which uncouples the electron transport system. Tubes 9/10 are inhibited by the presence of DNP which effects glucose transport as well as substrate entry into the electron transport system. Sodium malonate in tubes 11/12 is a competitive inhibitor of succinic dehydrogenase within the Krebs cycle. It will not halt the reactions of the ETS, and will only slow them down if glucose is in low concentration relative to the malonate. Finally, tubes 13/14 represent any endogenous reactions of the mitochondria in the sucrose isolation media.

Technically, glucose is not metabolized by the mitochondria. It must be altered by cytoplasmic enzymes to form pyruvate before entering the mitochondria. The isolation procedure used in mitochondrial preparation is important. If the mitochondria are "purified" the measurements will not work. Using a rough homogenate in sucrose will maintain enough enzymes for glycolysis to occur. The experiment could be altered by using pyruvic acid rather than the glucose. Glucose is used in the traditional experiment.

Record the fourteen manometer readings every 10 minutes for at least one hour. Average each pair of readings and substract the reading for 13/14 from each of the averages for 3/4, 5/6, 7/8, 9/10 and 11/12. Plot the corrected values against time, using a linear regression plot for each.

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