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Handout6 - Experiment leading to the Chemiosmotic Theory...

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Experiment leading to the Chemiosmotic Theory Isolate Mitochondria and add: Oxidizable Substrate (succinate), ADP, Pi Cellular Respiration: a. If oxygen is being consumed, the ETC is running b. If ATP synthesis is occurring (ATP is made), ATP Synthase is active At t = 4, add oligomycin (inhibitor of ATP Synthase). Now, the flow of H+ back into the matrix is blocked. The proton gradient becomes exceedingly large because the ETC continues to pump H+ into the intermembrane space. At some point, it will take too much energy to pump protons against the gradient into the intermembrane space, causing the ETC to stop. Thus at this point, O 2 consumption stops! At t = 8, add DNP (uncoupler). This is a hydrophobic, weak acid that diffuses into the matrix. Once in the matrix, the high pH causes H+ to dissociate, destroying the proton gradient. The removal of the gradient allows the ETC to start working again and consume O 2 once again (although ATP Synthase remains inhibited so no ATP is made) Uncoupling causes the ETC to work and O2 consumption to continue again! Shuttle Systems: These shuttle systems are needed to transport the 2 NADH made in the cytoplasm during Glycolysis into the mitochondrial matrix so that the electrons can be released at the ETC. (The electrons attached to NADH are not freely diffusible across the mitochondria membrane). The mechanism by which the 2 NADH are shuttled in to the matrix determines whether a total of 30 or 32 ATP will be made from a single glucose molecule. Two shuttle systems: 1. Malate-Aspartate Shuttle System: functions more in kidney, liver and heart tissue (pg. 731 in 5 th ed. textbook)
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NADH diffuses freely through the outer mitochondrial membrane, but is impermeable across inner membrane. Thus the electrons are transferred to oxaloacetate to form malate. Malate is then able to cross the inner membrane through a specialized malate-alpha-ketoglutarate transporter. In the matrix, malate is reconverted to oxaloacetate. Oxaloacetate, with the ammonia from group from
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