Studies on Isolated Mitochondria

1
Studies on Isolated Mitochondria
2
Objectives

• Isolate mitochondria
• Obtain respiration on a substrate
• Produce oxidative phosphorylation in vitro
• Explore the electron transport system (ETS)
• Evaluate the suitability of isolated mitochondria
  for further study

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Homogenate Composition
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Nuclear Pellet
Mitochondria, membrane vesicles, etc. in
supernatant
Nuclei and tissue fragments in pellet (nuclear
pellet)
5
Mitochondria Pellet
Without very high speed (ultra- centrifuge),
membrane vesicles remain in the supernatant
High speed brings down mitochondria
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Krebs Reactions in Vitro
( ) negligible concentration
Krebs cycle
(malate)
fumarate
Complex II
FADH2
succinate
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Electron Transport (NADH)
Matrix side
Respiration on NADH
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Succinate Pathway
Matrix side
Respiration on Succinate
9
Sites of Free Energy Change
Matrix side
Greatest free energy drop is associated with
three complexes
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Proton Translocation
H
H
H
Mandatory transport of H complexes I, III,
and IV
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Gradient Limits Respiration
H
H
H
H
H
H
H
X
H
H
X
X
H
H
H
H
X
H
X
X
X
No proton translocation means NO electron
transport
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State IV Respiration
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Mitochondria exhibit respiratory control
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State III Respiration
H
H
H
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ADPO Ratio (NADH)
H
H
H
Matrix side
All three proton translocation sites are active
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ADPO Ratio (Succinate)
H
H
Matrix side
Only two proton translocation sites are active
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ETS Inhibition
cyt c
Q
I
III
IV
II