Respiration in Yeast and Mammalian Cells
http://www.etsu.edu/biology/symbiosis/index.htm
Respiration in Yeast and Mammalian Cells
C6H12O6 + 6O2 +6H2O 6CO2 +12H2O
36 ATP/glucose molecule
Respiration: The cellular process by which glucose is oxidized to carbon dioxide and water. An inorganic molecule, often oxygen, serves as the final electron acceptor.The formation of ATP from this process can subsequently be used as the cell’s main source of energy
3 Step Process:1-Glycolysis2-TCA cycle3-Electron transport chain
Glucose
http://www.biologycorner.com/resources/resp_summary.jpg
Glycolysis
http://fig.cox.miami.edu/~cmallery/150/makeatp/sumgly.jpg
http://www.langara.bc.ca/biology/mario/Biol1116notes/biol1116chap6.html
Fermentation:
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookGlyc.html
http://www.langara.bc.ca/biology/mario/Biol1116notes/biol1116chap6.html
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookGlyc.html
http://mgl.scripps.edu/people/goodsell/pdb/pdb72/pdb72_1.html
F0F1 ATP synthase:
Matrix
Inner membrane
Inner membrane space
ADP + Pi ATP
H+ H+H+
Use mitochondria isolated from rat liver cells
Day 2:
Day 1:
Use cultures of Sacchromyces cerevisiae
Glucose
Pyruvate
Acetyl CoA
Acetaldehyde Ethanol
Electron Transport
Glucose and Ethanol Utilization in Yeast
NEM
Azide
Experiment 1: Kinetics of respiration in fresh yeast
Experiment 2: Kinetics of respiration in starved yeast
Experiment 3: Effects of NaN3 on respiration in starved yeast
Experiment 4: Effects of NEM on respiration in starved yeast
Day 1
•Dissolved oxygen combines with platinum electrons + protons to form water•Electrons move away from the silver electrode; positively charged silverions combine with chloride ions to form silver chloride•The remaining potassium ions combines with the hydroxyl ions from water
The more oxygen there is, the faster the electrons move away from the silver electrode and the more current that is produced
Its very important to keep the stirrer moving!
Teflon membrane
Sample chamber
KClElectrodePlatinum (-)
Silver (+)
The Clark Electrode:
P/O ratio=ATP synthesized/O2 utilized
AB
C
D
E
F
+m
ed
ium
+m
itos
+substrate
+ADP
Fraction of O2 used
CaCl2
0.4
0.8
1
time in minutes1 2 3 4
>3 indicates tightly coupledmitochondria
Day 2:
You will be testing various combinations of substrates, stimulators, inhibitors, and uncouplers on the rate of respiration
Antimycin A
Glutamate,malate-complex ISuccinate-complex IIAscorbate + TMPD-complex IV
Azide
OligomycinRotenone
http://www.cgm.cnrs-gif.fr/podospora/images/chaine_respi400.jpg
Many of the chemicals you will be using are toxic
Be Careful!!!!