Abstract
Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.
Highlights
Metabolic control analysis was applied to intact HepG2 cells
metabolic control analysis (MCA) has been used to estimate, both in isolated mitochondria and intact cells, the control coefficient of each respiratory complex; a value that has been shown to vary depending on tissues and on metabolic conditions leading to distinct mitochondrial energization states [9, 10]
We have shown that in intact cells, where the ATP synthase is not impaired by inhibitors, the metabolic control exerted by cytochrome c oxidase (CcOX) over the respiratory chain depends on ⌬
Summary
MCA has been used to estimate, both in isolated mitochondria and intact cells, the control coefficient of each respiratory complex; a value that has been shown to vary depending on tissues and on metabolic conditions leading to distinct mitochondrial energization states [9, 10]. Piccoli et al [11] have recently addressed the role of ⌬Hϩ on the control coefficient of CcOX (CIV), performing the investigation under nonphosphorylating conditions, i.e. in state 4 They found a low CIV value in the presence of either oligomycin, blocking ATP synthase, or oligomycin plus valinomycin, converting ⌬ into ⌬pH. We have extended previous studies in actively respiring HepG2 cells to further investigate the relative effect of ⌬ and ⌬pH on the control coefficient of CcOX, but under phosphorylating state 3 conditions [11], i.e. in the absence of oligomycin. Our results suggest that the depressing effect over respiration of ⌬Hϩ affects the distribution of control among the individual steps of the oxidative phosphorylation system
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