1. Effect of ADP on respiration.

- Adding more ADP increases the amount of oxygen consumed, consuming a certain amount of oxygen before leveling off again and stopping. Amount of oxygen consumed is proportional to amount of ADP added. - Calculate moles of ADP added to the solution, divide that by moles of oxygen consumed to work out an ADP:O ratio. - If NADH (supplied by adding glutamate) is used as substrate, ADP:O ratio is 2.5. This is in line with the fact that one NADH will lead to the production of 2.5 ATPs. 2. Effect of substrate. Using succinate instead of glutamate means FADH2 is provided rather than NADH. FADH2 contributes 1.5 ATPs because it feeds in later in the transport chain so the ADP:O ratio should be 1.5 instead. 3. Effect of uncouplers. DNP acts as an ionopore across the inner mitochondrial membrane. This lets the protons back out, defeating the proton gradient. This means lots of oxidative phosphorylation without creation of ATP. This means that the cell will never stop consuming oxygen. 4. MPP+ This stops oxygen consumption. It basically just blocks the electron transport chain. However, oxygen consumption resumes if succinate is added. This is because it inhibits complex 1, which is where NADH feeds. FADH2 feeds lower down so succinate is unaffected. 5. Cyanide Stops oxygen consumption for succinate and glutamate. Not an uncoupler as oxygen consumption is stopped. Since it stops both, it must inhibit complex III or IV.