If you are measuring oxygen consumption by isolated mitochondria, and you are in state 3 ADP stimulated respiration, will addition of substrate (say, pyruvate or glutamate) cause an increase in oxygen consumption?
In other words, is demand (represented by [ADP]) always limiting, or are there situations in which substrate is limiting?
Most in vitro experiments seem to use saturating amounts of ADP and or substrates, but the textbooks also say that in vivo mitochondria are sometimes in state 3 and sometimes in state 4 respiration. Occasionally we even see a humorous reference to "state 3.5" respiration.
In some ways the electron transport chain can be thought of one big enzymatically catalyzed reaction with multiple reactants and multiple products. Forty years ago, I learned that the rate of this reaction was controlled by the availability of ADP, but today I'm wondering if adding substrate sometimes increases oxygen consumption by mass action.
I'd value your thoughts. Thanks.
Daniel, thanks for this really helpful answer. Fig. 1 of the Li paper shows exactly what you describe; it's the kind of graph I spent half of yesterday looking for. It's a nice coincidence that I learned the notion that ADP was limiting while I was a grad student in physiology at Michigan in the 1970s. A couple years later I was a new assistant professor at Hopkins and learned of this paper:
https://www.ncbi.nlm.nih.gov/pubmed/7061429
which cemented that view of respiratory control. As a computer modeler, I was also influenced by the Kohn, Achs, and Garfinkel model of energy metabolism in rat heart.
Your reply and the Li paper you cited are convincing and I appreciate your taking the time to share your thoughts.
https://www.ncbi.nlm.nih.gov/pubmed/7061429
https://www.ncbi.nlm.nih.gov/pubmed/224719
I would like to point out that in our experiments (Li paper) all substrates are initially limited. That's because we permeabilize the cells (fibers), and substrates as well as ADP are washed out. When you add succinate to glutamate/malate/ADP stimulated mitos, you sure see an increase in respiration, b/c electrons are now also provided to complex II in addition to complex I (supplied through glut/mal), which further increases respiration.
Stephanie, Thanks for your comments. My understanding of the idea that ADP is normally limiting in vivo is that the proton gradient is thought to be at its maximum value in all/most physiological steady states. So in this case, electrons continue to flow to keep the proton gradient at that value even though ADP and Pi cause a steady flux of protons through FoF1. I think Fig 1 of your paper tells us that in a situation where the IMM has not yet been completely re-polarized after permeabilization, adding a complex 2 substrate can increase OCR significantly. Does that make sense to you?
It would be interesting to know whether succinate can increase OCR if glutamate/malate have been given sufficient time to return the proton gradient to its maximum value. I'm thinking succinate will have no effect if the IMM is already fully polarized. Rob
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