Talk:MiPNet12.15 RespiratoryStates
From Bioblast
State4 vs oligomycin
- Gyorgy Szabadkai (MD, PhD; London, UK) wrote:We have recently started to work on isolated mouse cardiac mitochondria on an Oroboros setup and would like to pick your brains on some puzzling results, see
attached. Below is a short description of the protocols used. State 4 respiration is very high, despite the very low uncoupled respiration when oligomycin is applied. What do we miss here?Cardiac mitochondria were isolated from a single mouse heart (0.1mg) using trypsin digestion (5mg/ml) and differential centrifugation. Fatty acid free solutions were used to avoid uncoupling mitochondrial respiration. All steps in the isolation were carried out at 4°C.Respiration was measured using an Oroboros respirometer at 37°C.
1) ADP titration0.25mg mitochondrial homogenate was added to a 2ml total volume (using miro 5 as respiration medium). Substrates used were pyruvate 10mM and malate 10mM. ADP 0.125mM added to achieve a State 3 respiration. After a steady State 4 respiration was achieved a further ADP 0.125mM was added. Max ADP (0.5mM) was finally added to check for saturation of ATPase at 0.125mM and subsequently oligomycin 2.5µM was added to assess Leak rate.
2) Effect of oligomycin on state 4 respirationAs before 0.25mg mitochondrial homogenate used. Oligomycin 2.5µM added after State 4 respiration achieved. We would highly appreciate your comments on these findings.
