Hello everybody,

 

 

My question is this: Imagine that you guys had a mitochondria(s) and a recently new discovered compound. How would you do to see if this new compound acts within the mitochondria? How you would know if this compound is, for example, inhibiting the electron transport chain? or destroying mitochondrial membranes? How to see if the compound acts or not in the mitochondria.

 

Thanks

Hi Raphael,

 

One possibility is to measure Oxygen consumption:

 

'Oxygen Electrode Measurement

 

Two independent Clark oxygen electrodes (Instech Laboratories, Plymouth Meeting, PA) with two independent thermojacketed chambers were used to analyze two samples in parallel. HeLa cells stably expressing the wt or the D255A form of p75 were incubated or not with 1 μM staurosporine for 6 hr as indicated. Cells were then digitonin permeabilized (see Supplemental Data) and washed with respiration buffer (RB = 250 mM sucrose, 2 mM EDTA, 30 mM KH2PO4, 5 mM MgCl2, 50 mM Tris [pH 7.4]) In Figures 3Dc and 3Dd, RB contained 2 mM ADP. The permeabilized cells were injected into the chambers containing 600 μl of air-saturated RB prewarmed at 37°C. To rule out an effect of dilution of cytochrome c, all measurements of apoptotic cells were performed in the presence of 100 μM cytochrome c. Substrates and inhibitors were added in the following order and final concentration: 5 mM malate, 5 mM glutamate, 2 μM rotenone, 5 mM succinate, 1 μM antimycin A. Oxygen concentration was calibrated with air-saturated buffer, assuming 390 ng atoms of oxygen/ml of buffer (Schulze-Osthoff et al., 1992). Rates of oxygen consumption are expressed as ng atoms of oxygen/min/mg of proteins.'

 

The overall morphology of the mitochondria would also be indicative and staining for cytochrome C release from the mitochondria would give a readout of mitochondrial outer membrane permeabilisation (MOMP). In order to confirm localisation of the compound at mitochondria, a mitochondrial fractionation may be performed, followed by detection of the compound in cytosolic versus mitochondrial fractions.

 

 

 

Hope this helps

 

Best wishes,

 

Tridimity

 

 

Ref.

 

Ricci J, Munoz-Pinedo C, Fitzgerald P, Bailly-Maitre B, Perkins GA, Yadava N, Scheffler IE, Ellisman MH & Green DR (2004) Disruption of Mitochondrial Function during Apoptosis Is Mediated by Caspase Cleavage of the p75 Subunit of Complex I of the Electron Transport Chain. Cell 117: 773-786

Edited July 10, 201312 yr by Tridimity