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Talk:MiPNet12.15 RespiratoryStates

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Discussion from the Mitochondrial Interest Group


State 4 vs oligomycin

  • Gyorgy Szabadkai (MD, PhD; London, UK): 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 screenshot of traces:
Szabadkai State4-vs-oligo.JPG

State 4 respiration is very high, despite the very low uncoupled respiration when oligomycin is applied. What do we miss here?

Below is a short description of the protocols used: 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 titration: 0.25mg 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 respiration: As before 0.25mg mitochondrial homogenate used. Oligomycin 2.5µM added after State 4 respiration achieved.

We would highly appreciate your comments on these findings.

Answers from MiG-List members

  • Marc Liesa Roig (PhD, Boston, MA): Higher state 4 than oligomycin is usually caused by the presence of ATPase activity from contaminant fractions in your crude mitochondrial fraction. This ATPase activity will generate a steady concentration of ADP from the ATP synthetised during state 3, thus increasing respiration. Therefore, determining respiration associated to proton leak using oligomycin would be the best option in these mitochondrial preparations.


  • Alvaro Elorza (PhD, Santiago, Chile): Just taking a quick look to your data, it seems you are running out of oxygen. That is why your oligomycin induced State 4 might have lower respiration.


  • Morten Scheibye-Knudsen (MD, Baltimore, MD): Both are probably true although contamination may contribute more. You can see an initial peak after you close the lid and before addition of ADP that is probably due to contamination with another ADP source + ATPase activity. In addition, after the first addition of ADP you never return to the pre-ADP state-4 indicating that the prep is most certainly contaminated with ATPases. The steady state respiration after your first ADP addition is therefore not a true state-4. I would look at optimizing the protocol and possibly test the cleanliness of your preps for example with a western.





Miro 5 Ingredients Concentration
EGTA 0.5mM
MgCl2.6 H2O 3mM
K-lactobionate 60mM
Taurine 20mM
KH2PO4 10mM
HEPES 20mM
Sucrose 110mM
BSA, essentially fatty acid free

Looks like perfectly-suited medium to stimulate the ATPases (various) in broken mitochondria and other junk co-isolated with heart mitochondria. I suggest you to make a basic medium composed e.g., of of 200mM mannitol, 70mM sucrose, 4 mM KH2PO4, 10mM MOPS (or HEPES) (adjust pH with KOH to 7.2-7.4), 0.2 mg/ml BSA fatty acids free, 0.5mM EGTA. Add the substrates (e.g., pyruvate + malate 5:2mM or succinate:glutamate 5:1mM), 30µM Diadenosine pentaphosphate (Ap5A, adenylate kinase inhibitor), and use 2µM carboxyatractylate to induce permanent “State 4” (before or after ADP addition). If this works for you and produces the desirable results, you can further complicate the matters by adding all those potassium, magnesium, taurine, lactobionate, whatever.

If it does not work for you, check your isolation procedure. E.g., you may decide to get rid of trypsin and use classical Chance&Hagihara’s 1960 B.Subtilis Nagarse protease (1 mg/ml; “subtilisin Carlsberg” I believe it is called these days) instead of it; or get rid of proteases altogether.


  • Christos Chinopoulos (MD, PhD, Budapest, Hungary): Omitting Mg2+ from the medium would definitely render contaminant ATPases inoperable, but I don't think this is a good idea; extramitochondrial Mg2+ concentration exerts a natural control on the ANT (PMID: 19289073), as it transports only the Mg2+ - (and H+)-free forms of ATP and ADP. In fact, ANT activity depends on extramitochondrial Mg2+ concentration, but this will NOT be reflected on your oxygen consumption curves, provided that you use sufficiently high amounts of ADP to saturate the Km of ANT for the adenine nucleotide. If in the future you wish to correlate your oxygen consumption data with ATP efflux data (PMID: 19289073) or any other ANT-mediated function, you would rather have Mg2+ in the medium.


Inhibiting adenylate kinase by Ap5A as Anatoly mentioned would also definitely help, but not enough in your case, if your contaminant ATPases from heart (mostly myosin ATPase type II and N/K ATPase) is an issue here, they both have extremely high activities. If you wish to refrain from using berrylium salts and orthovanadate because of disposal issues, you can use blebbistatin and ouabain to inhibit myosin ATPase type II and N/K ATPase, respectively (one more expensive than the other).


  • John J. Lemasters (MD, PhD, Charleston, SC): I have to disagree a little. Because the adenine nucleotide translocator transports ADP and ATP, not MgADP or MgATP, there is remarkably little effect of Mg-free medium on state 3 and 4 respiration and, specifically, on ADP-stimulated respiration. ATP synthase continues to work well. ATP synthase requires matrix Mg, but this Mg remains entrapped in the matrix during incubations in Mg-free medium, at least for the short term used for measurements of respiration.

Also in Mg-free medium, adenylate kinase is fully inhibited and diadenosine phosphate is not needed.


  • Christos Chinopoulos (MD, PhD, Budapest, Hungary): Dear John, you are absolutely right, but this is exactly what I have written "In fact, ANT activity depends on extramitochondrial Mg2+ concentration, but this will NOT be reflected on your oxygen consumption curves, provided that you use sufficiently high amounts of ADP to saturate the Km of ANT for the adenine nucleotide. " I was only referring to ATP efflux rates, NOT oxygen consumption.


  • Anatoly A. Starkov (PhD, New York, USA): I have also to disagree a little bit - whereas in Mg2+ -free medium adenylate kinase is truly fully inhibited, it is not so in a medium that was not rendered. Mg2+-free by artificial means (e.g., by adding EDTA as John have suggested initially). As I did not suggest adding EDTA - and still do not suggest doing so - a little bit (~ 30µM) of Ap5A would certainly not hurt and may even help.


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