一种由腺嘌呤核苷酸转运体介导的线粒体ATP-ADP交换率的新型动力学检测方法。
A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT.
作者信息
Chinopoulos Christos, Vajda Szilvia, Csanády László, Mándi Miklós, Mathe Katalin, Adam-Vizi Vera
机构信息
Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary.
出版信息
Biophys J. 2009 Mar 18;96(6):2490-504. doi: 10.1016/j.bpj.2008.12.3915.
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.
开发了一种利用ADP和ATP对Mg(2+)的不同亲和力的新方法来测量线粒体ADP-ATP交换率。向有活力的线粒体中添加ADP后，培养基中出现ATP的速率，是根据Mg(2+)敏感荧光指示剂Magnesium Green的膜不可渗透的5K(+)盐报告的线粒体外游离[Mg(2+)]的测量变化速率，使用标准结合方程计算得出的。该测定法的设计使得腺嘌呤核苷酸转位酶（ANT）是线粒体外体积中[Mg(2+)]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流出速率与膜电位函数关系的数据。最后，通过比较大鼠脑突触、脑非突触、心脏和肝脏线粒体中ATP-ADP稳态交换率与ANT的量，我们提供了已知ANT同种型的分子周转数。
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