Electron transfer-pathway state will depend on the substrate added to fuel the mitochondrial respiration. To obtain the NADH electron transfer-pathway state (N), NADH-linked substrates (CI-linked) are added, feeding electrons into the N-junction catalyzed by various mt-dehydrogenases. The most commonly applied N-junction substrate combinations are: PM, GM, PGM. The Succinate pathway control state (S), succinate-linked respiration or S-pathway is achieved with succinate as the single substrate. It is recommended to investigate the S-linked respiration in the presence of rotenone (Rot; or amytal, piericidine), which prevents accumulation of oxaloacetate. Oxaloacetate is a potent inhibitor of Complex II (CII; see succinate dehydrogenase, SDH). After inhibition of Complex I by rotenone, the NADH-linked dehydrogenases become inhibited by the redox shift from NAD+ to NADH. SDH is activated by succinate and ATP, which explains in part the time-dependent increase of respiration in isolated mitochondria after addition of rotenone (first), succinate and ADP.
- The following substrates can also be applied in this protocol:
- Glycerophosphate (Gp) which feeds the Q-junction
- Fatty acids to obtain the Fatty acid oxidation pathway control state
