Dufour S, Rousse N, Canioni P, Diolez P
Résonance Magnétique des Systèmes Biologiques, Bordeaux, France.
Biochem J. 1996 Mar 15;314 ( Pt 3)(Pt 3):743-51. doi: 10.1042/bj3140743.
The effects of temperature on the control of respiration rate, phosphorylation rate, proton leakage rate, the protonmotive force and the effective ATP/O ratio were determined in isolated rat liver mitochondria over a range of respiratory conditions by applying top-down elasticity and control analyses. Simultaneous measurements of membrane potential, oxidation and phosphorylation rates were performed under various ATP turnover rates, ranging from state 4 to state 3. Although the activities of the three subsystems decreased with temperature (over 30-fold between 37 and 4 degrees C), the effective ATP/O ratio exhibited a maximum at 25 degrees C, far below the physiological value. Top-down elasticity analysis revealed that maximal membrane potential was maintained over the range of temperature studied, and that the proton leakage rate was considerably reduced at 4 degrees C. These results definitely rule out a possible uncoupling of mitochondria at low temperature. At 4 degrees C, the decrease in ATP/O ratio is explained by the relative decrease in phosphorylation processes revealed by the decrease in depolarization after ADP addition [Diolez and Moreau (1985) Biochim. Biophys. Acta 806, 56-63]. The change in depolarization between 37 and 25 degrees C was too small to explain the decrease in ATP/O ratio. This result is best explained by the changes in the elasticity of proton leakage to membrane potential between 37 and 25 degrees C, leading to a higher leak rate at 37 degrees C for the same value of membrane potential. Top-down control analysis showed that despite the important changes in activities of the three subsystems between 37 and 25 degrees C, the patterns of the control distribution are very similar. However, a different pattern was obtained at 4 degrees C under all phosphorylating conditions. Surprisingly, control by the proton leakage subsystem was almost unchanged, although both control patterns by substrate oxidation and phosphorylation subsystems were affected at 4 degrees C. In comparison with results for 25 and 37 degrees C, at 4 degrees C there was evidence for increased control by the phosphorylation subsystem over both fluxes of oxidation and phosphorylation as well as on the ATP/O ratio when the system is close to state 3. However, the pattern of control coefficients as a function of mitochondrial activity also showed enhanced control exerted by the substrate oxidation subsystem under all intermediate conditions. These results suggest that passive membrane permeability to protons is not involved in the effect of temperature on the control of oxidative phosphorylation.
通过应用自上而下的弹性分析和控制分析，在一系列呼吸条件下，测定了温度对分离的大鼠肝脏线粒体呼吸速率、磷酸化速率、质子泄漏速率、质子动力势和有效ATP/O比值的影响。在从状态4到状态3的各种ATP周转率下，同时测量膜电位、氧化和磷酸化速率。尽管三个子系统的活性随温度降低（在37℃和4℃之间下降超过30倍），但有效ATP/O比值在25℃时出现最大值，远低于生理值。自上而下的弹性分析表明，在所研究的温度范围内，最大膜电位得以维持，并且在4℃时质子泄漏速率显著降低。这些结果明确排除了线粒体在低温下可能发生解偶联的情况。在4℃时，ATP/O比值的降低是由于添加ADP后去极化减少所揭示的磷酸化过程的相对减少[Diolez和Moreau（1985年），《生物化学与生物物理学学报》806，56 - 63]。37℃和25℃之间去极化的变化太小，无法解释ATP/O比值的降低。这一结果最好由37℃和25℃之间质子泄漏对膜电位的弹性变化来解释，即在相同膜电位值下，37℃时泄漏速率更高。自上而下的控制分析表明，尽管37℃和25℃之间三个子系统的活性发生了重要变化，但控制分布模式非常相似。然而，在4℃的所有磷酸化条件下获得了不同的模式。令人惊讶的是，尽管在4℃时底物氧化和磷酸化子系统的控制模式均受到影响，但质子泄漏子系统的控制几乎未变。与25℃和37℃的结果相比，在4℃时，当系统接近状态3时，有证据表明磷酸化子系统对氧化和磷酸化通量以及ATP/O比值的控制增加。然而，控制系数作为线粒体活性函数的模式也表明，在所有中间条件下，底物氧化子系统的控制作用增强。这些结果表明，质子的被动膜通透性不参与温度对氧化磷酸化控制的影响。
