Brand M D, Chen C H, Lehninger A L
J Biol Chem. 1976 Feb 25;251(4):968-74.
We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxtbutyrate were accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis.
我们以琥珀酸作为呼吸底物，添加鱼藤酮阻断NAD连接的电子传递，研究了大鼠肝线粒体对Ca2+的能量依赖性摄取。在存在3-羟基丁酸或其他可渗透的一元羧酸的情况下，Ca2+的摄取程度接近在存在磷酸盐时观察到的程度。通过绘制3-羟基丁酸摄取量与Ca2+摄取量的关系图的斜率来确定阳离子和阴离子摄取之间的定量关系，该方法无需对早期非能量化或非化学计量结合事件进行模糊校正即可确定化学计量。该程序表明，每一个Ca2+离子积累2个3-羟基丁酸分子。在这些条件下，呼吸链每个能量保存位点每对电子积累近2个Ca2+离子和4个3-羟基丁酸分子。由于3-羟基丁酸必须质子化才能以未解离的游离酸形式通过膜，因此得出结论，呼吸链每个能量保存位点每对电子排出（随后重新吸收）4个质子，这与化学渗透假说假设的值2.0相反。
