Stoner C D
J Biol Chem. 1987 Aug 5;262(22):10445-53.
P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.
通过直接测定含有己糖激酶捕获剂的牛心线粒体悬浮液中的无机磷酸（Pi）摄取量，估算了跨越呼吸链不同部分的六个测定系统中的P/2e化学计量比。电子供体为苹果酸+丙酮酸、琥珀酸以及抗坏血酸+N,N,N',N'-四甲基对苯二胺，电子受体为铁氰化物（位点1、位点2以及位点1 + 2）和氧气（位点1 + 2 + 3、位点2 + 3以及位点3）。一个主要目标是找到这样的条件，即这六个系统产生的结果具有足够好的一致性，以便对其可靠性有信心。这个目标实现了，并且分别在位点1、位点2和位点3系统中观察到最大值为1.1、0.5和1.0。这要求能量守恒反应相对不构成限制，并且P/2e比值应根据通过呼吸链抑制剂抑制氧化磷酸化所获得的呼吸速率与磷酸化速率的关系图的斜率来估算。后一个要求可以避免明显的内源性解偶联剂的影响，并且基于这样的观察结果（邹承鲁和范达姆，1969年，生物化学与生物物理学报172卷，174 - 176页），即低浓度的解偶联剂在低呼吸速率下降低磷酸化速率的绝对量几乎与在高呼吸速率下一样多。位点1处的最大P/2e化学计量比被认为是1.0，位点1系统中观察到的值被认为由于在转氢酶水平上质子排出而更高。研究了因羧基苍术苷抑制而产生的呼吸控制，发现各系统之间差异很大。有人指出，这一观察结果与缺乏完全控制主要归因于离子循环不一致，鉴于此，位点3处相对较弱的控制与氧气在偶联膜基质侧被还原不一致。
