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-muH) was investigated. The overall O2 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 (CIV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (CIV = 0.85) was also measured in the presence of nigericin, i.e., when Delta-muH is maximal, and in the presence of nigericin and valinomycin (CIV = 0.77), when Delta-muH is abolished. In contrast, CcOX displayed a markedly lower control coefficient (CIV = 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.
Control of respiration by cytochrome c oxidase in intact cells: role of the membrane potential
2009
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-muH) was investigated. The overall O2 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 (CIV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (CIV = 0.85) was also measured in the presence of nigericin, i.e., when Delta-muH is maximal, and in the presence of nigericin and valinomycin (CIV = 0.77), when Delta-muH is abolished. In contrast, CcOX displayed a markedly lower control coefficient (CIV = 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
