BIOC 212 Lecture Notes - Lecture 62: Atp Synthase, Secretion, Oligomycin

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Metabolism XXIV
Real Biochemistry
All of the material we have learned has been studied using controlled
For example, inhibitors of different ETC complexes were used to show what the
order of the chain was
o Complexes reduced upon treatment were determined to be upstream of the
inhibitor’s target
o Look at which complex becomes reduced when add inhibitor compared to
Complexes that are downstream of the inhibitor become fully oxidized
Complexes that are upstream will become fully reduced
Blockage of electron transit at some point
Have a set of tools that allows us to block these different enzymes in various
ways; by putting together results from all these experiments, were able to
generate chain of electron transit
Mitochondria Isolation
Mitochondria can be isolated & separated biochemically
Palade was first to isolate mitochondria from tissue
o Invented a lot of the electron microscopy techniques used to study
organelles and subcellular compartments
o Mitochondrial import pathway
Mitochondria have 2 membranes so are pretty resistant to lysis
o Can lyse the membrane to lyse open the cell and spill out the cytosol
o Spin down components
Largest = nuclei
Next largest = mitochondria
o Can re-suspend the pellet and spin it down a little slower; get rid of nuclei
Spin it down faster, and collect just intact mitochondria
Can add metabolites or ions and see how levels affect mt transport, signalling,
Can also try to purify the different components of the mitochondria
o Get rid of outer membrane
Relatively easy to do since is much more porous and water permeable
than inner membrane
Place mitochondria in pure water --all salt that is inside the matrix will
cause the matrix to swell
Outer membrane will pop due to tension from expanding matrix
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