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
Glycolysis
• Occurs in the cytosol
Hi [ATP]
• 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
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
1
O
2 2
1
FADH2 + O2
2
NADH + H+ +
NAD+ + H2O
FAD + H2O
B. Phosphorylation step
ADP + Pi
ATP
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
Oxidative
Phosphorylation
• 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
Summary
Glucose
ATP
Fig. 16-9
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]
Applicable to yeast
respiration, not purified
mitochondria—why?
N-ethylmaleimide
Fig. 16-3
Yeast ethanol metabolism
EtOH
ADH
acetaldehyde
Glucose
ATP
acetic acid
CoA
Electron transport chain
inhibitors and substrates
rotenone
Antimycin A
Sodium azide
Ascorbate + TMPD
Glutamate, malate
Fig. 16-19
Inhibitors and uncouplers of
oxidative phosphorylation
Inhibitors
•
Atractyloside: ADP/ATP antiporter
•
Oligomycin:ATP synthase
Atractyloside
oligomycin
Uncouplers
•
•
DNP shuttles H+ across inner
membrane, dissipates gradient
DNP
CaCl2 stimulates oxidative
phosphorylation and ATP production
Ca2+
Fig. 16-32
Summary of Cellular Energetics
Glucose
High [ATP]
(Pasteur effect)
Glycolysis
N-ethylmaleimide
Pyruvate
NADH
Malate
FADH2
EtOH
Acetyl CoA
Citric Acid Cycle
Succinate
Fig. 16-2
Uncouplers
Ca+2, DNP
NADH + FADH2
Rotenone
Antimycin A
Ascorbate + TMPD
Sodium Azide
Electron transport chain
Energy released used to pump H+ creating
an elecrochemical gradient
Flow of protons down the gradient fuels ATP synthase
Atractyloside
ADP + Pi
O2
Oligomycin
ATP
H2O
Oxidative
Phosphorylation
Carbon Dioxide Emission Control Authority
Review:
Characterization of Cellular Components

Who?

What?

Where?

When?

How?

Why?
Review

Immunofluorescence microscopy



Vital staining



Microscope
Cell staining
Colocalization
Filters
Transfection


Eukaryotic expression vectors
GFP