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Biophys J. 2009 Mar 18;96(6):2490-504. doi: 10.1016/j.bpj.2008.12.3915.

A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT.

Author information

1
Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary.

Abstract

A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.

PMID:
19289073
PMCID:
PMC2907717
DOI:
10.1016/j.bpj.2008.12.3915
[Indexed for MEDLINE]
Free PMC Article
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