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

C6H12O6 + 6 O2 + 36 Pi +36 ADP + 36 H+

6 CO2 + 36 ATP + 42 H2O

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. 16-3

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

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

Step 3: Electron transport chain and oxidative phosphorylation

Oxidative Phosphorylation

Oxidative phosphorylation is the process by which the energy stored in NADH and FADH2 is used to produce ATP.

A. Oxidation step: electron transport chain

B. Phosphorylation step

NADH + H+ + O2NAD+ + H2O

12

FADH2 + O212

FAD + H2O

ADP + Pi ATP

Electron Transport Chain

Fig. 16-19

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

Fig. 16-32

• 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. 16-9

SummaryGlucose

ATP

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

Inhibitors of Glycolysis

Hi [ATP]

Fig. 16-3

N-ethylmaleimide

Applicable to yeast respiration, not purified mitochondria—why?

ADH

acetaldehyde

EtOH

acetic acid CoA

Yeast ethanol metabolism

Glucose

ATP

Electron transport chaininhibitors and substrates

Fig. 16-19

rotenone Antimycin A

Sodium azide

Glutamate, malate

Ascorbate + TMPD

Fig. 16-32

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+

Inhibitors and uncouplers of oxidative phosphorylation

Summary of Cellular EnergeticsGlucose

Pyruvate

Acetyl CoA

NADH + FADH2

Electron transport chain

O2 H2OEnergy released used to pump H+ creating an elecrochemical gradient

Flow of protons down the gradient fuels ATP synthase

ADP + Pi ATP

Glycolysis

Citric Acid Cycle

Oxidative Phosphorylation

N-ethylmaleimide

EtOH

Succinate

MalateFADH2

NADH

RotenoneAntimycin A

Sodium Azide

UncouplersCa+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