1. MAIN

The page contains many links that lead outside of semmelweis.hu addresses. Please use the page and follow other links at your own risk. Contact Dr. Christos Chinopoulos for inquiries and more information. 

The present website outlines the methodology for measuring mitochondrial ADP-ATP exchange rate mediated by the adenine nucleotide translocase (ANT),  originally published in: Biophys J. 2009 Mar 18;96(6):2490-504. doi: 10.1016/j.bpj.2008.12.3915, entitled “A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT” by Christos Chinopoulos, Szilvia Vajda, László Csanády, Miklós Mándi, Katalin Mathe and Vera Adam-Vizi.

The method can be applied not only in isolated mitochondria, but also permeabilized cells, tissue homogenates and ANT expressed in liposomes.
An improved calibration step of this method has been published in Methods Enzymol. 2014;542:333-48.

Follow the numbered tabs. The ‘core’ of the method is outlined in the Buffers, chemicals, Kds determination of ADP, ATP to Mg2+, and Conversion of free Mg2+ to ATP. However, it is advisable to go through all of the links, if you wish to understand what you will be actually doing.

Here you can also find a link pointing to work related to ANT1 as a voltage sensor of the mitochondrial permeability transition.

 

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2. Mitochondria: adenine nucleotide transport basics

3. Current method: concept conceived by others

4. Other methods: problems with luciferase

5. Affinity of ions to ADP, ATP

6. Why Magnesium Green?

7. Buffers, chemicals

8. Kds determination of ADP, ATP to Mg2+

9. Conversion of free Mg2+ to ATP

10. Dependence of ANT activity to Mg2+

11. Dependence of ANT activity on DΨm

12. Dependence of ANT activity on pH

13. Influence of other parameters on ANT activity

14. Limitations of the method

15. 'Under the hood' advices

16. 'Over the hood' advices

17. More tricks: determination of ATP consumption rate and ATP/ADP ratio

18. Erev_ANT, Erev_ATPase

19. Erev estimator

20. Erev modeler

21. What can you do with this method?

22. CREDITS

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