Template:SUIT text D033

SUIT-020 Fluo mt D033 is a protocol to assess the additivity between N-pathway and S- pathway in the Q-junction as well as investigate the N- and NS-pathway control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be assessed by the Omy titration test. Addition of PM (Pyruvate & Malate) to the mitochondria leads to the hyperpolarisation of the mt-membrane, while ADP (D) decreases the mt-membrane potential. Glutamate (G) and Succinate (S) are able to further increase the mt-membrane potential. Rotenone, which is an inhibitor of Complex I, depolarises the mt-membrane. Addition of Omy results in hyperpolarisation owing to the fact that the inhibition of the ATP synthase leads to accumulation of protons in the intermembrane space. Uncoupler depolarises the mt-membrane in a concentration-dependent manner, therefore leading to dissipation of the mt-membrane potential. Complex III inhibitor Antimycin A (Ama) blocks the respiration and dissipates the mt-membrane potential. For mt-membrane potential analysis and calculation, visit this page: Mitochondrial membrane potential.