I saw that in most protocols- when measuring respiration (of isolated mitochondria, using the Seahorse), both pyruvate+ malate are added.
I was wondering what would be the result of adding only the pyruvate? shouldn't it be a sufficient substrate to generate both oxaloacetate and AcCoA?
Thanks!
Hi Chen,
I have never worked with Seahorse, but in principle, if you put just pyruvate, as the oxaloacetate concentration inside mitochondria is too low, you will not be able to drive Krebs Cycle properly. So, most people use pyr + mal or glutamate + mal to ensure that they can drive Krebs cycle properly and generates NADH+ enought to test maximal complex I respiration-linked.
But, do not use too much malate, since high malate concentrations can inhibit SDH and some complexes from ETS.
Best,
Henver
Thanks!
But- isn't only a low concentration of OAA needed? and what about pyruvate carboxylase that can generate OAA from pyruvate?
The conversion of malate to OAA produces one NADH+, which is good for total activation of complex I. Also, the convertion of pyruvate to OAA needs ATP and produces ADP in the reaction, if you have this, mitochondrial membrane potential will decrease and complex I activity will increase to keep the mitochondrial membrane potential, so you will have a factor that you can`t control.
Best,
Henver
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