BIOC 212 Lecture Notes - Lecture 62: Atp Synthase, Secretion, Oligomycin
29 May 2018
School
Department
Course
Professor
Metabolism XXIV
Real Biochemistry
• All of the material we have learned has been studied using controlled
experiments
• 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
control
• 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,
etc.
• 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
