Talk:MiPNet17.06 IOC66
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IOC66 Workshop experiments
2012-03-15 First Demo Experiment
- Oxygraph EF (in both chambers the same SUIT-Protocol was applied)
- SUIT-Protocol: S + D5 + H2O2 + H2O2 + F
- the homogenate was added as well as Amr and HRP
- addition of S (without rotenone) for LEAK respiration (without ADP) - succinate is the substrate for succinate dehydroganse (CII) resulting in succinate oxidation and accumulation of oxaloacetat (a stronger inhibitor of succinate dehydrogenase than malonate) that can not permeate the mitochondrial inner membrane - inhibition of CII
- reverse electron flow from CII to CI is known to stimulate production of ROS species under these experimental conditions - represented by the increase of the fluorescence signal
- addition of ADP inhibited respiration approx. 77 to 95% - drop down of the flux and the fluorescence signal
- no stimulation because of the inhibtion of CII by the accumulation of oxaloacetat (without rotenone: succinate - fumarat - malat - oxaloacetat that accumulates and inhibits CII)?
- addition of rotenone: S(Rot) supports electron flux exclusively through CII; inhibition of the NADH-linked dehydrogenases through inhibition of NADH oxidation preventing accumulation of oxaloacetate
- addition of FCCP in the end: further inhibition of respiration and no increase of the oxygen flux (with FCCP the oxygen flux should increase due to a higher oxygen consumption) - hint: mitochondria already in an uncoupled state?
2012-03-16 Second Demo Experiment
- SUIT-Protocol (chamber E): S + Amp/HRP + D0.15 + D5 + Omy + F + G + H2O2 + c
- addition of small amounts of ADP (0.15 mM) already inhibits respiration in the presence of succinat - 33% inhibition
- additional ADP (5 mM) inhibits respiration again - 36% inhibition (69% in total)
- oligomycin (omy; for the evaluation of leak state in the presence of ADP ): inhibition of ATP synthase and oxyen flux should be reduced; in the biological experiment the oxygen flux increased after addition of omy - omy also acts as an uncoupler!
- a rise in the oxygen concentration (reoxygenation with pure oxygen) increased the flux after titration of omy whereas the titration of FCCP diminished the oxygen flux - no further uncoupling occured
- addition of glutamate as CI-linked substrate increased the oxygen flux
- prominent cyt c effect (51%) - quality of preparation? duration of the protocol - late application of cyt c?
- SUIT-Protocol (chamber F): Amp/HRP + S + G + D0.15 + D5 + Omy + F + H2O2 + c
- addition of succinate and glutamate for CI/CII-Leak respiration without ADP
- titration of 0.15 mM ADP stimulated respiration (55%) whereas addition of 5 mM ADP inhibited respiration about 22% - but this inhibibition could be due to oxygen limitation (no steady state reached!)
- reoxgenation with pure oxygen increased the oxygen flux - similar to oxygen flux with 0.15 mM ADP and steady state!
- oligomycin reduced the oxygen flux as well as FCCP - mitochondria dyscoupled?
- high cytochrome c effect (64.7%)
Notes
- mitochondria uncoupled/dyscoupled already from the beginning?
- investigation on CI is necessary (NADH)
- conversion of ADP to ATP?
- stimulation of respiration with succinate and glutamate - elimination of oxaloacetate through transamination?
- P/E ratio > 1: ETS is less than OXPHOS capacity - experimental artefact (inhibitors of ATP synthase suppress ETS capacity, particularly in stressed cells)
- cyt c effect: damage of the outer mitochondrial membrane
- when should the addition of cyt c occur to evaluate the effect?
- do not add cyt c in a Leak state - leads to activation of respiration unrelated to injury of the outer mitochondrial membrane
- add rotenone together with succinate - rotenone inhibits CI by inhibition of NADH oxidation
- huge cytochrome c effect as there was not a big stimulation of CI-linked respiration and also the titration of succinate did not really stimulate respiration
