Wanders R J, Van den Berg G B, Tager J M
Biochim Biophys Acta. 1984 Oct 26;767(1):113-9. doi: 10.1016/0005-2728(84)90085-9.
The values reported in the literature for the extramitochondrial ATP/ADP ratio in resting rat-liver mitochondria (State 4) vary widely. The conditions required for an accurate determination of this parameter were therefore investigated. In experiments with rat-liver mitochondria incubated under State-4 conditions, it was found that the extramitochondrial ATP/ADP ratio, as calculated from the values measured in neutralised perchloric acid extracts, was lower than that estimated from the concentrations of creatine and creatine phosphate, using the metabolite indicator method. The discrepancy is due to hydrolysis of ATP occurring in the presence of perchloric acid. Conditions are described for minimising ATP hydrolysis in the presence of perchloric acid, and include the use of low concentrations of perchloric acid, short times of exposure to the acid before neutralisation, low temperatures and the presence of excess EDTA. Under these conditions, the values obtained for the extramitochondrial ATP/ADP ratio agreed with those calculated by the metabolite indicator method, provided ratios do not exceed the value of 100. In cases where the extramitochondrial ATP/ADP does exceed 100, phenol/chloroform/isoamyl alcohol must be used to quench the reactions, as described by Slater et al. (Slater, E.C., Rosing, J. and Mol, A. (1973) Biochim. Biophys. Acta 292, 534-553). With this method, the extramitochondrial ATP/ADP ratio was found to have a value of more than 1000 in rat-liver mitochondria incubated with succinate + rotenone in the resting state (pH 7.0; T = 37 degrees C), in agreement with Slater et al.
文献报道的静息大鼠肝脏线粒体（状态4）线粒体外ATP/ADP比值差异很大。因此，对准确测定该参数所需的条件进行了研究。在用处于状态4条件下孵育的大鼠肝脏线粒体进行的实验中，发现从中性化高氯酸提取物中测得的值计算出的线粒体外ATP/ADP比值，低于使用代谢物指示法根据肌酸和磷酸肌酸浓度估算的值。这种差异是由于高氯酸存在下ATP发生水解所致。描述了在高氯酸存在下使ATP水解最小化的条件，包括使用低浓度的高氯酸、中和前短时间接触酸、低温以及存在过量的乙二胺四乙酸（EDTA）。在这些条件下，只要比值不超过100，测得的线粒体外ATP/ADP比值与代谢物指示法计算的值一致。在 线粒体外ATP/ADP超过100的情况下，必须如斯莱特等人所述（斯莱特，E.C.，罗辛，J.和莫尔，A.（1973年）《生物化学与生物物理学报》292，534 - 553），使用苯酚/氯仿/异戊醇淬灭反应。用这种方法，发现在静息状态（pH 7.0；温度 = 37摄氏度）下用琥珀酸 + 鱼藤酮孵育的大鼠肝脏线粒体中，线粒体外ATP/ADP比值超过1000，这与斯莱特等人的结果一致。
