A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells
Mitochondrial ADP, ATP Translocases
We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg(2+)] reported by an Mg(2+)-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg(2+). In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF(3)(-) and sodium orthovanadate (Na(3)VO(4)) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F(1)F(o)-ATPase due to its sensitivity to BeF(3)(-) and Na(3)VO(4). With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.