Marijke J. Wagner

Selectivity of TMC207 towards Mycobacterial ATP Synthase Compared with That towards the Eukaryotic Homologue

ABSTRACT The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC50] of >200 μM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC50 of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.

Regulation of thermogenesis in flowering Araceae: The role of the alternative oxidase

The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.

Immunological identification of the alternative oxidase of Acanthamoeba castellanii mitochondria

Mitochondria of the protozoa Acanthamoeba castellanii possess a cyanide‐insensitive oxidase cross‐reacting with monoclonal antibodies raised against the plant alternative oxidase. Immunoblotting revealed three monomeric forms (38, 35, and 32 kDa) and very low amounts of a single 65 kDa dimeric form. Cross‐linking studies suggest that while in plants the alternative oxidase occurs as a dimer, in amoeba it functions as a monomer. Immunologically detectable protein levels change with the age of amoeba cell culture. Increased amounts of the 35 kDa protein are accompanied by an increase in the activity of cyanide‐resistant respiration.

Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.

Measurements of in Vivo Ubiquinone Reduction Levels in Plant Cells.

A method is described for the determination of in vivo ubiquinone (UQ) reduction levels in nongreen tissues by extraction and subsequent detection of ubiquinone-10 and ubiquinol-10 with high-performance liquid chromatography. In Petunia hybrida cell suspensions UQ reduction remained at a stable level of about 60%, despite the changing conditions during the batch culture (from excess sugar to starvation) and the concomitant variations in respiration. Also, in the presence of uncoupler, which causes a large increase in respiration via both the cytochrome pathway and the alternative pathway, UQ reduction levels stayed at 60%. In mitochondria isolated from these cells, activity of the alternative pathway was only observed at UQ reduction levels higher than 80%. It is proposed that in vivo the relationship between UQ reduction and the activity of the alternative oxidase is modulated by mechanisms such as thiol modifications and accumulation of organic acids. Accordingly, pyruvate concentration in P. hybrida cells increased in the presence of uncoupler.

Effect of oxygen on growth and respiration of potato tuber callus

Growth and oxygen uptake of potato callus is faster in an oxygen-enriched atmosphere (70% oxygen, v/v; “oxygen-callus”) than in air (20% oxygen, v/v; “air-callus”). Especially the non-mitochondrial, so-called ‘residual respiration’ is increased in “oxygen-callus”. The capacities of the mitochondrial respiratory pathways (cytochrome pathway, Vcyt and alternative pathway, Valt) are also higher in this callus. In both callus types only a small part of the alternative pathway capacity is used in uninhibited respiration. The lower oxygen uptake of “air-callus” at normal air oxygen pressures is partially due to diffusional impedance. Measurement of the respiratory parameters of “air-callus” in oxygen-saturated medium leads to higher values than measurement in air-saturated medium, although these values are still lower than those of “oxygen-callus”.ATP-production was calculated from the oxygen-uptake data and compared with the dry weight production of the callus to give values of 10.0 and 10.8 g dry weight produced.-mol ATP-1, for “air-callus” and “oxygen-callus” respectively. As no harmful side-effects are observed, cultivation of callus under elevated oxygen pressures may be useful, when rapid callus-growth is necessary.

Modular kinetic analysis reveals differences in Cd2+ and Cu2+ ion-induced impairment of oxidative phosphorylation in liver.

Impaired mitochondrial function contributes to copper‐ and cadmium‐induced cellular dysfunction. In this study, we used modular kinetic analysis and metabolic control analysis to assess how Cd2+ and Cu2+ ions affect the kinetics and control of oxidative phosphorylation in isolated rat liver mitochondria. For the analysis, the system was modularized in two ways: (a) respiratory chain, phosphorylation and proton leak; and (b) coenzyme Q reduction and oxidation, with the membrane potential (Δψ) and fraction of reduced coenzyme Q as the connecting intermediate, respectively. Modular kinetic analysis results indicate that both Cd2+ and Cu2+ ions inhibited the respiratory chain downstream of coenzyme Q. Moreover, Cu2+, but not Cd2+ ions stimulated proton leak kinetics at high Δψ values. Further analysis showed that this difference can be explained by Cu2+ ion‐induced production of reactive oxygen species and membrane lipid peroxidation. In agreement with modular kinetic analysis data, metabolic control analysis showed that Cd2+ and Cu2+ ions increased control of the respiratory and phosphorylation flux by the respiratory chain module (mainly because of an increase in the control exerted by cytochrome bc1 and cytochrome c oxidase), decreased control by the phosphorylation module and increased negative control of the phosphorylation flux by the proton leak module. In summary, we showed that there is a subtle difference in the mode of action of Cd2+ and Cu2+ ions on the mitochondrial function, which is related to the ability of Cu2+ ions to induce reactive oxygen species production and lipid peroxidation.

The mitochondrial outer membrane is not a major diffusion barrier for ADP in mouse heart skinned fibre bundles.

The response of mitochondrial oxygen consumption to ADP in saponin-skinned cardiac fibre bundles has an apparent Km an order of magnitude higher than that in isolated mitochondria. Here we report that incubating skinned cardiac fibre bundles from wild-type mice or double-knockout mice lacking both cytosolic and mitochondrial creatine kinase (CK) with CK and creatine or with yeast hexokinase and glucose as extramitochondrial ADP-producing systems decreases the apparent Km of the bundles for ADP severalfold. We conclude that the affinity of mitochondria for ADP in mouse heart is of the same order of magnitude as that of isolated mitochondria, while the high apparent Km of the bundles is caused by diffusion gradients outside the mitochondria.

Changes in alternative oxidase activity and other mitochondrial parameters with callus formation by potato tuber tissue discs

Abstract Incubation of potato tuber tissus discs on agar with hormones (kinetin, KN and naphthyleneacetic acid, NAA) leads to callus induction which doubles the fresh weight in approx. 3 weeks. Changes during this incubation in some characteristics of mitochondrial isolated from this tissue have been compared with those isolated from tissue placed on agar without hormones. A considerable increase in mitochondrial state III respiration is found with both treatments. Antimycin-A resistant respiration (alternative oxidase activity) which is not detectable in freshly-sliced tissue, increases during the first days of incubation on the agar. It stabilises then at a level which is 3–4 times higher in callus-forming tissue than in tissue placed on agar without hormones. Cytochrome oxidase activity of mitochondria from callus forming tissue increases steadily during the first 3 weeks of callus induction. In mitochondria from tissue, not treated with hormones, cytochrome oxidase activity increases only during the first days and then stabilises. The increase in alternative oxidase activity (expressed as a percentage of state III respiration) in mitochondria from tissue placed on agar without hormones is greater when older tubers are used as a source for the potato tuber tissue. Finally, after approx. 10 months of storage of the tubers, this relative increase equals that of callus-forming tissue. Experiments with tubers during the first months after harvest indicate that the higher level of alternative oxidase in mitochondria from callus-forming tissue in this period, might be caused by the presence of the auxin in the agar.

Low affinity membrane transporters can increase net substrate uptake rate by reducing efflux

Cells require membrane-located transporter proteins to import nutrients from the environment. Many organisms have several similar transporters for the same nutrient, which differ in their affinity. Typically, high affinity transporters are expressed when substrate is scarce and low affinity ones when substrate is more abundant. The benefit of using low affinity transporters when high affinity ones are available has so far remained unclear. Here, we investigate two hypotheses. First, it was previously hypothesized that a trade-off between the affinity and the maximal catalytic rate explains this phenomenon. We find some theoretical and experimental support for this hypothesis, but no conclusive evidence. Secondly, we propose a new hypothesis: for uptake by facilitated diffusion, at saturating extracellular substrate concentrations, lowering the affinity enhances the net uptake rate by reducing the substrate efflux rate. As a consequence, there exists an optimal, external substrate concentration dependent transporter affinity. An in silico analysis of glycolysis in Saccharomyces cerevisiae shows that using the low affinity HXT3 transporter instead of the high affinity HXT6 enhances the steady-state flux by 36%. We tried to test this hypothesis using yeast strains expressing a single glucose transporter that was modified to have either a high or a low affinity. Due to the intimate and reciprocal link between glucose perception and metabolism, direct experimental proof for this hypothesis remained inconclusive in our hands. Still, our theoretical results provide a novel reason for the presence of low affinity transport systems which might have more general implications for enzyme catalyzed conversions.