Kesseler A, Diolez P, Brinkmann K, Brand M D
Universität Bonn, Abteilung für experimentelle Okologie, Federal Republic of Germany.
Eur J Biochem. 1992 Dec 15;210(3):775-84. doi: 10.1111/j.1432-1033.1992.tb17480.x.
Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.
采用代谢控制分析的自上而下方法，测定了纯化的马铃薯线粒体对氧化磷酸化的控制。在从静止（状态4）到最大（状态3）的一系列磷酸化速率范围内，测量了呼吸链、磷酸化反应和质子泄漏对呼吸速率、磷酸化速率、质子泄漏速率和质子动力势的控制。这些速率是通过在葡萄糖存在下添加不同量的己糖激酶，或在ADP存在下添加不同量的寡霉素获得的。呼吸底物是NADH或琥珀酸，两者都直接将电子传递给泛醌。以NADH为底物时，交替氧化酶途径的耗氧速率可忽略不计，但以琥珀酸为底物时可测量，并已减去。除了接近状态4时质子泄漏的控制同样重要或更重要外，在所有速率下，马铃薯线粒体中呼吸速率的控制主要由呼吸链施加。对于NADH的氧化，在状态3下呼吸链对呼吸速率的通量控制系数在0.8至1.0之间，而在状态4下，呼吸速率的控制在呼吸链和质子泄漏之间大致平均分配。磷酸化速率的控制主要由呼吸链施加，尽管在低速率下磷酸化系统的控制也很重要。对于NADH的氧化，在状态3下呼吸链对磷酸化速率的通量控制系数为0.8 - 1.0，而在接近状态4时，磷酸化系统对磷酸化速率的通量控制系数约为0.8，呼吸链为0.25。质子泄漏速率的控制由呼吸链和质子泄漏共同分担；磷酸化系统具有负控制。对于NADH的氧化，在状态3下泄漏对泄漏速率的通量控制系数为1.0，呼吸链为0.4，磷酸化系统为 - 0.4，而在状态4下泄漏速率的通量控制系数泄漏约为0.5，呼吸链为0.5。对质子动力势大小的控制很小，在 - 0.2至 + 0.2之间，这反映了系统保持质子动力势相当恒定的运作方式；呼吸链和磷酸化系统具有相等且相反的控制，除了接近状态4时质子泄漏的控制非常小。
