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 C 6 H 12 O O P i +36 ADP + 36 H + 6 CO ATP + 42 H 2 O

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

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

Hi [ATP] Fig Glycolysis 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

Step 2: Citric Acid Cycle

Mitochondria

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, FADH 2, NADH, FADH 2 enter the electron transport chain

Step 3: Electron transport chain and oxidative phosphorylation

Oxidative Phosphorylation Oxidative phosphorylation is the process by which the energy stored in NADH and FADH 2 is used to produce ATP. A. Oxidation step: electron transport chain B. Phosphorylation step NADH + H + + O 2 NAD + + H 2 O 1 2 FADH 2 + O FAD + H 2 O ADP + P i ATP

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

Fig 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 Oxidative Phosphorylation

Fig Summary Glucose ATP

This week’s lab Day one: Yeast respiration Goal: learn how to measure O 2 consumption Compare O 2 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

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

ADH acetaldehyde EtOH acetic acid CoA Yeast ethanol metabolism Glucose ATP

Electron transport chain inhibitors and substrates Fig rotenone Antimycin A Sodium azide Glutamate, malate Ascorbate + TMPD

Fig Inhibitors Atractyloside: ADP/ATP antiporter Oligomycin:ATP synthase Uncouplers DNP shuttles H + across inner membrane, dissipates gradient CaCl 2 stimulates oxidative phosphorylation and ATP production Atractyloside oligomycin DNP Ca 2+ Inhibitors and uncouplers of oxidative phosphorylation

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

Carbon Dioxide Emission Control Authority

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

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