The sum of flux control coefficients in the electron-transport chain of mitochondria.

The sum of the flux control coefficients for group-transfer reactions such as electron transport has been proposed to be two when the coefficients are calculated from experiments in which the concentrations of the electron carriers are changed (CE) but one when they are calculated from changes in the rates of the electron-transfer processes (Cv). We tested this proposal using electron transport in uncoupled beef heart, potato tuber and rat liver mitochondria. First, with ascorbate plus N,N,N',N"-tetramethyl-p-phenylenediamine as substrate, the CE flux control coefficients of ascorbate, N,N,N',N"-tetramethyl-p-phenylenediamine, mitochondria and oxygen over electron-transport rate were measured by direct titration of the concentrations of these electron carriers. CE values were close to zero, one, one and zero, respectively, giving a sum of CE flux control coefficients of approximately two. At higher concentrations of N,N,N',N'-tetramethyl-p-phenylenediamine, its CE control decreased and the sum decreased towards one as predicted. Secondly, the Cv control coefficients of groups of electron-transfer processes with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine as substrate were measured. This was achieved by measuring the effects of KCN (or malonate or N,N,N',N'-tetramethyl-p-phenylenediamine) on system flux when intermediates were allowed to relax and on local flux when intermediates were held constant. The Cv flux control coefficients were calculated as the ratio of the effects on system flux and on local flux. The sum of the Cv flux control coefficients was approximately one. Whether a sum of one or a sum of two was obtained depended entirely on the definition of control coefficients that was used, since either sum was obtained from the same set of data depending on the method of calculation. Both definitions are valid, but they give different information. It is important to be aware of which definition is being used when analysing control coefficients in electron-transport chains and other group-transfer systems.

Localisation of the sites of action of cadmium on oxidative phosphorylation in potato tuber mitochondria using top-down elasticity analysis.

The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely protonmotive force. The three subsystems were substrate oxidation, which produces protonmotive force, and the proton leak reactions and the phosphorylation reactions, which consume protonmotive force. By measuring the effect of cadmium on the kinetic response of each subsystem to protonmotive force (top-down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of protonmotive force and by diverting proton flux from phosphorylation reactions to the proton leak reactions.

Effects of cadmium on the control and internal regulation of oxidative phosphorylation in potato tuber mitochondria.

The effect of cadmium on the distribution of control over oxidative phosphorylation in potato tuber mitochondria was quantified by measuring control coefficients using top-down metabolic control analysis. Oxidative phosphorylation was divided into three subsystems, namely substrate oxidation, the phosphorylation reactions and the proton leak. The control exerted by each of these subsystems over the system fluxes, the value of the protonmotive force and the effective P/O ratio was quantified in the presence of different concentrations of free cadmium (up to 21 microM). Cadmium is known to stimulate the proton leak and inhibit the substrate oxidation reactions, but it had little effect on the distribution of control over the system variables except to shift the pattern to lower rates. Control exerted by particular subsystems appeared to change or to stay the same as cadmium was varied, depending on whether the control coefficients were presented as a function of respiration rate or protonmotive force. The regulatory strength of protonmotive force on the system variables was also calculated, as partial internal response coefficients. These coefficients changed with ATP turnover rate and with cadmium concentration, showing how the internal regulation of oxidative phosphorylation shifts under different conditions. The values of control coefficients and partial internal response coefficients show where control lies and how intermediates regulate the system variables under different conditions of ATP demand and external effector (i.e. cadmium) concentration. However, they are not useful for identifying the sites of action of external effectors, for which elasticity and regulation analysis must be used.

Quantitative determination of the regulation of oxidative phosphorylation by cadmium in potato tuber mitochondria.

The effects of cadmium on respiration rate, phosphorylation rate, proton leak rate, the protonmotive force and the effective P/O ratio were determined over a range of respiratory conditions and cadmium concentrations by applying top-down regulation analysis. To quantify the effects of cadmium, we determined the overall response coefficients of these variables of oxidative phosphorylation to cadmium in different respiratory states between state 4 and state 3 and at different cadmium concentrations. The overall response coefficients to cadmium showed quantitatively how cadmium stimulated substrate oxidation rate at high cadmium concentrations near state 4 but inhibited it to different extents under all other conditions, how cadmium inhibited the rate of proton leak rate at low cadmium concentrations near state 4 but stimulated it under all other conditions, and how cadmium inhibited the rate of phosphorylation and depressed the protonmotive force and the effective P/O ratio to different extents under all conditions. Cadmium is known to stimulate the proton leak and to inhibit the substrate oxidation reactions; we calculated the elasticities of these subsystems to cadmium to quantify its effects. To describe fully how the cadmium effects on different subsystems produce the overall responses of the system to cadmium, we then calculated the partial response coefficients of the system variables to cadmium acting through each subsystem. The partial response coefficients quantify the contribution of each block to the overall effect of cadmium on each variable in any condition, and sum to the overall response coefficient in each condition. Together with the elasticity analysis and the control analysis and internal regulation analysis presented in the preceding papers [Kesseler, A. & Brand, M. D. (1994) Eur. J. Biochem. 225, pp. 897-906; Kesseler, A. & Brand, M. D. (1994) Eur. J. Biochem. 225, pp. 907-922] they completely describe how cadmium exerts its effects on oxidative phosphorylation at the system level.

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

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.

[Arthropathies following the administration of pefloxacin to an adolescent with mucoviscidosis]. / Arthropathies consécutives à l'administration de péfloxacine chez un adolescent atteint de mucoviscidose.

We report the occurrence of knee joint effusion with prolonged functional impairment in a 17-year old boy with cystic fibrosis treated with pefloxacin for recurrent lower respiratory tract infections caused by Pseudomonas aeruginosa. Because the quinolone pefloxacin fairly often induces joint disease in pediatric age groups, we advocate restricting its use to those patients whose growth is completed. In growing individuals, it seems reasonable to use second generation quinolones only in infections caused by resistant organisms and to avoid exerting the joints during treatment.

[Double deficiency of sulfite and xanthine oxidase causing encephalopathy and due to a hereditary anomaly in the metabolism of molybdenum]. / Double déficit en sulfite et xanthine oxydase, cause d'encéphalopathie due à une anomalie héréditaire du métabolisme du molybdène.

The clinical features and biological results in a second patient with a metabolic defect of the molybdenum cofactor are described. The first case was reported in 1978 by Duran et al. Their clinical description was similar with early encephalopathy and myoclonial and dislocation of the lens. Biologically, this condition is characterised by secondary hypo-uricemia and hypo-uricuria due to xanthine oxidase deficiency and by sulphituria, resulting from sulphite oxidase deficiency. These two enzymes have a common hepatic molybdenum cofactor, the structure and metabolism of which are only partially known.