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Lab 6 - Cellular energetics

Lab 6 - Cellular energetics. Objective: to examine respiration in yeast and rat mitochondria. Techniques : Measure effects of substrates and inhibitors on oxygen consumption in yeast and rat mitochondria using an oxygen polarograph. ATP Synthesis and glucose metabolism.

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Lab 6 - Cellular energetics

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  1. Lab 6 - Cellular energetics Objective: to examine respiration in yeast and rat mitochondria Techniques: Measure effects of substrates and inhibitors on oxygen consumption in yeast and rat mitochondria using an oxygen polarograph

  2. ATP Synthesis and glucose metabolism C6H12O6 + 6 O2 + 36 Pi +36 ADP + 36 H+ 6 CO2 + 36 ATP + 42 H2O

  3. Overview of Cellular Respiration Images from Purves et al., Life: The Science of Biology, 4th Edition

  4. Step 1: Glycolysis Glucose + 2ADP 2 pyruvate + 2ATP

  5. Glycolysis Hi [ATP] • Occurs in the cytosol • Glucose metabolized to 2 pyruvate + 2 ATP • High [ATP] inhibits phosphofructokinase (PFK) • High [ADP] stimulates PFK • Pasteur Effect: Increase in the rate of carbohydrate breakdown that occurs when switched from aerobic to anaerobic conditions Fig. 16-3

  6. Step 2: Citric Acid Cycle

  7. Mitochondria

  8. Citric Acid Cycle

  9. Citric Acid Cycle • a.k.a. Krebs Cycle, TCA Cycle • Occurs in mitochondrial matrix • Pyruvate reacts with CoA to form Acetyl CoA • NAD+, FAD+ reduced to NADH, FADH2, • NADH, FADH2 enter the electron transport chain

  10. Step 3: Electron transport chain and oxidative phosphorylation

  11. 1 1 2 2 Oxidative phosphorylation is the process by which the energy stored in NADH and FADH2 is used to produce ATP. Oxidative Phosphorylation A. Oxidation step: electron transport chain NAD+ + H2O NADH + H+ + O2 FADH2 + O2 FAD + H2O B. Phosphorylation step ADP + Pi ATP

  12. Electron Transport Chain During electron transport, energy released is used to transport H+ across the inner mitochondrial membrane to create an electrochemical gradient Fig. 16-19

  13. OxidativePhosphorylation • H+ transport results in an electrochemical gradient • Proton motive force: energy released by flow of H+ down its gradient is used for ATP synthesis • ATP synthase: H+ channel that couples energy from H+ flow with ATP synthesis Fig. 16-32

  14. Summary Glucose ATP Fig. 16-9

  15. This week’s lab Day one: Yeast respiration • Goal: learn how to measure O2 consumption • Compare O2 consumption by normal and starved yeast Day two: Mitochondria • Examine the effects of various inhibitors and substrates on the rate of respiration • Determine the identity of your unknown (think what substrates you need to add and in what order together with the unknown

  16. Inhibitors of Glycolysis Hi [ATP] Applicable to yeast respiration, not purified mitochondria—why? N-ethylmaleimide Fig. 16-3

  17. Yeast ethanol metabolism EtOH ADH acetaldehyde Glucose CoA acetic acid ATP

  18. Electron transport chaininhibitors and substrates rotenone Antimycin A Sodium azide Ascorbate + TMPD Glutamate, malate Fig. 16-19

  19. Inhibitors and uncouplers of oxidative phosphorylation Inhibitors • Atractyloside: ADP/ATP antiporter • Oligomycin:ATP synthase Uncouplers • DNP shuttles H+ across inner membrane, dissipates gradient • CaCl2 stimulates oxidative phosphorylation and ATP production Atractyloside oligomycin DNP Ca2+ Fig. 16-32

  20. Summary of Cellular Energetics Glucose High [ATP] (Pasteur effect) Glycolysis N-ethylmaleimide Pyruvate EtOH Acetyl CoA NADH Malate Citric Acid Cycle FADH2 Succinate Fig. 16-2 Uncouplers Ca+2, DNP NADH + FADH2 Rotenone Antimycin A Electron transport chain Ascorbate + TMPD Sodium Azide Energy released used to pump H+ creating an elecrochemical gradient O2 H2O Oxidative Phosphorylation Flow of protons down the gradient fuels ATP synthase Oligomycin Atractyloside ADP + Pi ATP

  21. Carbon Dioxide Emission Control Authority

  22. Review:Characterization of Cellular Components • Who? • What? • Where? • When? • How? • Why?

  23. Review • Immunofluorescence microscopy • Microscope • Cell staining • Vital staining • Colocalization • Filters • Transfection • Eukaryotic expression vectors • GFP

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