It is a cardinal error to assume that increase in activity (of an enzyme or enzyme system) reflects increase in relevant proteins or vice versa. The entire emphasis on ex.phos has been tight controls that go far beyond protein levels by WB or whatever. SOD levels increase in muscle with exercise as also heat shock proteins

calliong for a different paradigm of explanation. So may be in your case or it may not.

Dear Erica, Dr. Sitaramam is absolutely right indicating error in considering that increase in one metabolic pathway must be accompanied by increase in another. There is, though, some sense in this proposition, but it is too mechanistic. Mitochondrial enzymes have, particularly those of Oxphos, redundancy because they have to function in a very large range in their activity. This is particularly relevant for the muscle tissues of the heart and especially skeletal muscle. In addition, you have to remember that production of superoxide radicals is most active when the organ is less active. I have also to add some criticism of your measurements of the TCA cycle. Measurements of separate enzymes of the TCA cycle provide very little information about activity of the cycle as a whole. All enzymes, in fact, work as one functional unit and also can work separately in active organs (brain, heart, sk, muscles) as two coupled cycles : from OAA ---> citrate synthase to a-ketoglutarate, and another from a-KG -->succinate to OAA. And the left cycle (a-KG-OAA) may have 5 fold higher activity than the right cycle. In some tissues (prostate) the right cycle may be totally inactive, producing only citrate in large quantities.

Hi Erica,

an increase in SOD2 might be the result of an increase in mitochondrial activity OR an increased mitochondrial stress, thus the need for the dismutase to convert the superoxide into H2O2. Checking this is easy: use mitosox to measure mitochondrial superoxide in your samples and compare them to control.

Good luck!

p.

Thank both of you for your comments, I completely agree.

This has made me reflect on the interpretation of the results and see them from a different perspective. I was wondering if it is possible to measure the activity of TCA cycle as a whole, is there any reliable way?

Thank you again for your time,

Erica

Erica, it seems to me that your understanding of the TCA cycle is wrong. In mitochondria nothing works alone. TCA cycle works with several metabolic pathways, both anabolic and catabolic, substrates come into and go in other pathways simultaneously. This is in vivo. In vitro, you MUST provide mitochondria with various substrates mixtures, and using incubation conditions optimal for the mitochondria, not just sucrose. This is a new paradigm I have been developing for a number of years. I have attached my latest paper published in EC Cardiology.

The activity of the TCA you can measure only through measuring respiratory activities. Most current measurements of separate enzymes or single substrates, and succinate + rotenone are obsolete.

Peter Bou Dib Thanks a lot for your suggestion! We are really considering to perform this assay.

Erica

Alexander V Panov thanks again for your paper, I am sure that it will be very useful to clarify some concepts.

Erica

Dear Erica,

in addition to what exactly reported by Dr Panov and Dr Sitamaran about TCA and OXPHOSP features, as well as their criticism to the wrong, but widely used, thought that an higher protein evel definitely means higher rate in a metabolic pathway, you should also consider the role played by mitochondrial shuttle/s which independently of TCA can transfer redox equivalents to respiratory chain.

Thus, any evaluation of L-Lactate/Pyruvate, glycerol3P and other working shuttles in your tissue wouldl be of some help.

Best

Gianluca

A very timely notion by Dr. Paventi. I can add only that shuttles work differently in different tissues, and also depend on the type of substrates used by the tissue (or researcher). Heart and brain, in particular, use mostly malate/aspartate shuttle.

To illustrate the fact that provision of a physiological mixture of substrates may give completely different results from the old single substrate method, I have attached a picture from my experiments< which was presented at some meeting ,but never published. The details are described in the attachments.