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!!!!