A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT
A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT
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DOI：
10.1016/j.bpj.2008.12.3915
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发表时间：
2009-03-18
影响因子：
3.4
通讯作者：
Adam-Vizi, Vera
中科院分区：
文献类型：
--
作者：
Chinopoulos, Christos;Vajda, Szilvia;Adam-Vizi, Vera
我们提出了一种利用ADP和ATP对Mg2+的差异亲和力来测量线粒体ADP-ATP交换率的新方法。在激活的线粒体中加入ADP后，培养基中出现ATP的速率是根据Mg2+敏感荧光指示剂镁绿的不透膜5K（+）盐报告的游离线粒体[Mg2+]的变化率计算的，使用标准结合方程。该试验的设计使得腺嘌呤核苷酸转位酶（ANT）是线粒体外体积中[Mg2+]变化的唯一介质，这是ADP-ATP交换的结果。我们还提供了在6.8-7.8基质pH范围内ATP外排率作为膜电位函数的依赖性数据。最后，通过比较ATP-ADP稳态交换速率与大鼠脑突触、脑非突触、心脏和肝脏线粒体中蚂蚁的数量，我们提供了已知蚂蚁同型的分子周转率。
A novel method exploiting the differential affinity of ADP and ATP to Mg2+ 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 [Mg2+) reported by the membrane-impermeable 5K(+) salt of the Mg2+-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 [Mg2+] 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.