Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption.
Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption.
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DOI：
10.1016/b978-0-12-416618-9.00017-0
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发表时间：
2014
影响因子：
--
通讯作者：
Starkov AA
中科院分区：
文献类型：
--
作者：
Chinopoulos C;Kiss G;Kawamata H;Starkov AA
我们之前已经描述了一种荧光法来测量通透性细胞线粒体中的ADP-ATP交换率，其中存在几种消耗大量ATP的酶和其他相互竞争的转换腺嘌呤核苷酸的反应。这种方法依赖于用镁离子敏感的荧光指示剂镁绿™记录线粒体外游离镁离子的变化，利用腺苷二磷酸和三磷酸腺苷对镁离子的不同亲和力。特别是在BeF_3、−和Na_3VO_4存在下，洋地黄素使细胞通透性增强，抑制了所有利用三磷酸腺苷和二磷酸腺苷的反应，但腺苷核苷酸转位酶催化了三磷酸腺苷与二磷酸腺苷的线粒体交换。然后，根据线粒体外游离镁离子的变化率，使用标准结合方程，计算出当ADP加入到有能量的线粒体时，在介质中出现的三磷酸腺苷的速率。在这里，我们描述了这种方法的一个变种，包括改进的校准步骤。这一步骤最大限度地减少了在将mGr™信号转换为线粒体外游离[mg2+]和atp的过程中可能引入的误差。此外，我们描述了一种将这种方法与同一样本中线粒体膜电位和氧气消耗的测量相结合的方法。本文描述的方法对于研究恶性细胞是有用的，众所周知，恶性细胞在低氧环境中茁壮成长，并具有深刻的功能变化的线粒体。
We have previously described a fluorometric method to measure ADP–ATP exchange rates in mitochondria of permeabilized cells, in which several enzymes that consume substantial amounts of ATP and other competing reactions interconverting adenine nucleotides are present. This method relies on recording changes in free extramitochondrial Mg2+ with the Mg2+-sensitive fluorescent indicator Magnesium Green (MgGr)™, exploiting the differential affinity of ADP and ATP for Mg2+. In particular, cells are permeabilized with digitonin in the presence of BeF3− and Na3VO4, inhibiting all ATP- and ADP-utilizing reactions but mitochondrial exchange of ATP with ADP catalyzed by the adenine nucleotide translocase. The rate of ATP appearing in the medium upon the addition of ADP to energized mitochondria is then calculated from the rate of change in free extramitochondrial Mg2+ using standard binding equations. Here, we describe a variant of this method involving an improved calibration step. This step minimizes errors that may be introduced during the conversion of the MgGr™ signal into free extramitochondrial [Mg2+] and ATP. Furthermore, we describe an approach for combining this methodology with the measurement of mitochondrial membrane potential and oxygen consumption in the same sample. The method described herein is useful for the study of malignant cells, which are known to thrive in hypoxic environments and to harbor mitochondria with profound functional alterations.