We have designed a novel family of drugs that target an enzyme within the mitochondrial matrix.
A number of those compounds have been shown to inhibit the target enzyme in cell-free assays. However, only one compound is active in cell-based assays.
We attribute this discrepancy to the fact that most compounds cannot freely access the mitochondrial matrix.
Which would me the easiest way to test this hypothesis? In other words, we would like to have a rough idea of the concentration of each compound in the mitochondrial matrix. It does not need to be very precise.
Your best bet might be to harvest and lyse the cells (mechanically), isolate the mitochondria (centrifugation), then perhaps lyse the mitochondria and assay for your drug.
Here's a pretty detailed protocol for isolating mitochondria from a variety of sources:
http://www.nature.com/nprot/journal/v2/n2/full/nprot.2006.478.html
Hello Peter,
Thanks for the answer. That was the first thing that came to my mind too. However, if we follow those procedures, would not we be detecting the drug from the inter-membrane space as well? We are interested in determining the concentration within the mitochondrial matrix only.
Best regards,
Miro
Following the links, I have found this:
http://www.ncbi.nlm.nih.gov/pubmed/422680
Hello Miro,
In any case previously it is necessary to use inhibitors of all type of transporter system to prevent the moving your drug during mitochondria isolation.
Beside that you can make a model experiment. Intact mitochondria after incubation at the different concentration of your drug must be treated by the inhibitors of all type of transporters and then quickly must be separated from medium with the high speed refrigerated centrifuge.
Eugene Maevsky
Thanks Eygene. It seems really tricky. It may be a good business opportunity for kit makers... ;)
Hi
You have to harvest cell, lysis the cell and isolate mitochondria then use western blotting methods or Eliza.
Hi Edelmiro,
I'm not a chemistry, but i think that you could add to your compound (and as control even to the others drugs that don't work) some specific sequence for a mitochondrial matrix enzyme. Then you could verify that only your drug is it inside the matrix because it will be the only that will give you the specific reaction. In this way you will demonstrate that only your compound is inside the matrix and not the other. Probably is not easy to do, but you could try. On the other hand, you wrote that your compound block an enzyme inside the matrix. You could evaluate some substrate of this enzyme and you should find a different amount ( phosphorylation- cleavage - i don't know what is your enzyme )compared to the untreated or the treated with the un-functional drugs.
i don't know if they are good ideas, but with so few informations i can't help more.
regards.
Francesco
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