Brand M D, Harper M E, Taylor H C
Department of Biochemistry, University of Cambridge, U.K.
Biochem J. 1993 May 1;291 ( Pt 3)(Pt 3):739-48. doi: 10.1042/bj2910739.
The control exerted by substrate oxidation reactions, by ATP turnover and by the proton leak over the oxygen consumption rate, the phosphorylation rate, the proton leak rate and the protonmotive force (delta p) in isolated rat liver mitochondria under a range of conditions between non-phosphorylating (State 4) and maximum phosphorylation (State 3) was investigated by using the top-down approach of metabolic control analysis. The experiments were carried out with saturating concentrations of the substrates succinate, glutamate with malate, or pyruvate with malate. The distribution of control was very similar with each of the three substrates. The effective P/O ratio (i.e. not corrected for leak reactions) was also measured; it varied from zero in State 4 to 80-90% of the maximum theoretical P/O ratio in State 3. Under most conditions control over the effective P/O ratio was shared between proton leak (which had negative control) and the phosphorylating subsystem (which had roughly equal positive control); near State 4, substrate oxidation reactions also acquired some control over this ratio. In resting hepatocytes the effective P/O ratio was only 50% of its maximum theoretical value, corresponding to an effective P/O ratio of only 1.3 for complete oxidation of glucose. The effective P/O ratio for intracellular mitochondrial oxygen consumption was 64% of the maximum value. The control coefficient of the mitochondrial proton leak over the effective P/O ratio in cells was -0.34; the control coefficient of phosphorylation reactions over this ratio was 0.31 and the control coefficient of substrate oxidation reactions over the ratio was 0.03, showing how the coupling efficiency in cells can respond sensitively to agents that change the proton leak or the ATP demand, but not to those that change substrate oxidation.
采用代谢控制分析的自上而下方法，研究了在非磷酸化（状态4）和最大磷酸化（状态3）之间的一系列条件下，底物氧化反应、ATP周转以及质子泄漏对分离的大鼠肝线粒体中氧消耗率、磷酸化率、质子泄漏率和质子动力势（Δp）的控制作用。实验使用了饱和浓度的底物琥珀酸、谷氨酸与苹果酸或丙酮酸与苹果酸。三种底物中的每一种的控制分布都非常相似。还测量了有效P/O比（即未校正泄漏反应）；其范围从状态4时的零到状态3时最大理论P/O比的80 - 90%。在大多数条件下，对有效P/O比的控制由质子泄漏（具有负控制）和磷酸化子系统（具有大致相等的正控制）共同分担；在接近状态4时，底物氧化反应也对该比例获得了一些控制。在静息肝细胞中，有效P/O比仅为其最大理论值的50%，对应于葡萄糖完全氧化时的有效P/O比仅为1.3。细胞内线粒体氧消耗的有效P/O比为最大值的64%。细胞中质子泄漏对有效P/O比的控制系数为 - 0.34；磷酸化反应对该比例的控制系数为0.31，底物氧化反应对该比例的控制系数为0.03，这表明细胞中的偶联效率如何能够灵敏地响应改变质子泄漏或ATP需求的试剂，但对改变底物氧化的试剂不敏感。
