* Vytautas Magnus University, Vileikos 8, 3035 Kaunas, Lithuania; and
Institute for Biomedical Research, Kaunas Medical University, Eiveniu 4, 3009 Kaunas, Lithuania
Received August 13, 2001; accepted October 15, 2001
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ABSTRACT |
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Key Words: 2,2`,5,5`-tetrachlorobiphenyl; liver mitochondria; oxidative phosphorylation; respiration; membrane potential; ATP synthase; Complex I; kinetic analysis.
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INTRODUCTION |
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An essential feature unifying many noxious effectors is their simultaneous interaction with many different sites within the oxidative phosphorylation system. The scheme of the oxidative phosphorylation system in mitochondria is presented in Figure 1. Electrons from oxidizable substrates enter the respiratory chain via different routes: electrons to Complex I (NADH dehydrogenase) are donated by the substrates reducing NAD+, while electrons originating from succinate are passed to ubiquinone (UQ) via Complex II (succinate dehydrogenase). Further, the electrons are transferred to oxygen via Complex III (cytochrome bc1 complex), cytochrome c, and Complex IV (cytochrome oxidase). Electron flow through Complexes I, III, and IV is coupled with the outward pumping of protons, producing both chemical (
pH) and electrical (
) gradient because the inner mitochondrial membrane has very low natural permeability to protons (the proton leak). The ATP synthase makes use of the electrochemical gradient by coupling the inward proton flux with the ADP phosphorylation reaction. In addition, transport of substrates for ATP synthesis (phosphate and ADP) into mitochondrial matrix is also driven by the membrane potential (
).
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MATERIALS AND METHODS |
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The quality of mitochondrial preparations was determined by the respiratory control index (RCI), equal to the ratio of the respiratory rates (V3/V2) of mitochondria in State 3 and in State 2, according to the common terminology (Chance and Williams, 1955).
The respiration and membrane potential of mitochondria were measured in a closed, stirred, and thermostated 1.5-ml vessel fitted both with a Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, UK) and a tetraphenylphosphonium (TPP+)-selective electrode (A. Zimkus, Vilnius University, Lithuania).
To maintain mitochondrial respiration in the stationary state, one of the requirements of modular kinetic, or "top-down," analysis (Fell, 1997; Hafner et al., 1990
), we used the creatine phosphokinase ADP-regenerating system, which was developed in our laboratory earlier (Kholodenko et al., 1987
) for the analysis of action of different agents affecting the processes involved in oxidative phosphorylation. The peculiarity of this system is that it does not exert control over respiration, and therefore the oxygen consumption rate is determined only by mitochondrial processes. This advantage allows optimum manifestation of changes in kinetics of oxidative phosphorylation induced by any effector. This experimental system has been successfully used for the investigation of effects of various effectors or conditions on the functional activity of mitochondria (Borutaite et al., 1989
, 1995
; Marcinkeviciute et al., 2000
, Mildaziene et al., 1995
, 1996
).
The experiments were performed at 37°C using 5 mM succinate (+ 2 µM rotenone) or 5 mM glutamate + 5 mM malate as a substrate. Mitochondrial concentration in the probe was 1.0 mg/ml. The rate of mitochondrial respiration corresponding to the rate in State 3 was registered after addition of 1 mM ATP to the incubation medium containing 110 mM KCl, 20 mM Tris-HCl, 5 mM KH2PO4, 50 mM creatine, excess of creatine kinase, 1 mM MgCl2, pH 7.2. TPP+ (133266 nM) was added for the membrane potential measurements. Mitochondrial swelling was monitored spectrophotometrically at 520 nm wavelength in the same medium.
Modular kinetic analysis was applied to determine the kinetic changes induced by 2,2`,5,5`-TCB on the level of the oxidative phosphorylation system. In this approach, we split the system of oxidative phosphorylation into three subsystems, or modules, connected by the proton motive force (p), which consists of two components: the mitochondrial membrane potential (
) and the proton concentration gradient (
pH).
p is generated by the respiratory subsystem (R) and consumed by the phosphorylation subsystem (P) and the membrane leak subsystem (L). We could measure changes in
p as changes in
without introducing a significant error, provided the
pH value was small and the changes in
pH were negligible. Direct measurement of
pH in liver mitochondria (Mildaziene et al., 2000
) confirmed that this was so in the case of our experiments. Therefore, we estimated activities of the subsystems from dependencies of the fluxes through these subsystems on
(Fig. 2
). We determined the following kinetic dependencies on
:
The dependence of the flux through the respiratory subsystem (JR) on is determined by titrating mitochondrial respiration in State 3 with an inhibitor of the phosphorylation system (carboxyatractyloside 00.15 nmol/mg mitochondrial protein).
The dependence of the flux through the membrane leak subsystem (JL) on is determined under the conditions of complete inhibition of phosphorylation by excess oligomycin (1 µg/mg). The rate of succinate oxidation is titrated with malonate (03 mM), and the rate of glutamate + malate oxidation with rotenone (00.06 nmol/mg mitochondrial protein).
The dependence of the flux through the phosphorylation subsystem (JP) on is determined using a similar protocol of titrations (with malonate, 01 mM; rotenone, 00.013 nmol/mg mitochondrial protein) under the conditions of active respiration (without oligomycin). JP at any given
is calculated as JP = JR JL at the same
.
The effect of 2,2`,5,5`-TCB on oxidative phosphorylation was estimated from these three titration sets, performed on the same preparation of liver mitochondria in the absence of 2,2`,5,5`-TCB and in the presence of 20 µM 2,2`,5,5`-TCB. We used 2,2`,5,5`-TCB (99.8% purity) manufactured by Chem Service, West Chester, PA.
The membrane potential generated during ATP hydrolysis was measured with TPP+-selective electrode in the same medium without a substrate and creatine kinase, and in the presence of antimycin A (2 µg/mg protein). In all assays, the functional parameters of mitochondria were monitored after a 3-min preincubation of mitochondria with 20 µM 2,2`,5,5`-TCB.
ATPase activity was measured at 37°C by recording the pH changes in the incubation chamber during the ATP hydrolysis reaction in medium containing 110 mM KCl, 2.5 mM Tris-HCl, 2.5mM KH2PO4, 5 mM MgCl2, antimycin A (140 ng/mg protein), 3 mM ATP, 70 nM carbonyl cyanid-m-chlorophenylhydrasone (CCCP), pH 8. Concentration of mitochondria in these experiments was 1.5 mg/ml. Buffering capacity of the reaction mixture was determined experimentally by recording the pH change induced by addition of the known quantity of HCl (12 nM H+) to the reaction mixture after each measurement. ATPase activity was estimated from the initial rate of increase in the H+ concentration during ATP hydrolysis. Control experiments showed that the H+ formation was inhibited by oligomycin by more than 95%.
Activity of Complex I was measured spectrophotometrically by following the kinetics of NADH reduction at 340 nm (Ragan et al, 1987) in fractured mitochondria (by rapid freezing-thawing of mitochondria, repeated four times). Measurements were performed at 37°C in medium containing 110 mM KCl, 20 mM Tris-HCl, 5 mM KH2PO4, 1 mM MgCl2, antimycin A (1 µg/ml), 0.1 mg/ml NADH, fractured mitochondria (0.2 mg mitochondrial protein/ml), pH 7.2. The reaction was started after a 3-min preincubation by adding 100 µg/ml coenzyme Q10. Enzymatic activity was calculated using the extinction coefficient of NADH 6.81 mM1cm1.
Statistical analysis.
The mean of each experiment was calculated as average for two or three repetitive runs. The effect of a toxic agent was analyzed on the same mitochondrial preparation by comparing their functional parameters in the absence of 2,2`,5,5`-TCB and in the presence of 20 µM 2,2`,5,5`-TCB (paired experiments). The data points in figures and text are expressed as the mean of 35 experiments on different preparations of mitochondria ± SE. Statistical analysis of the data was done by Student t-test. Statistical significance was assumed at p < 0.05.
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RESULTS |
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The results obtained in experiments with liver mitochondria oxidizing succinate + rotenone are presented in Figures 3A, 3B, and 3C. It can be seen that 2,2`,5,5`-TCB decreased the rate of mitochondrial respiration in State 3 from 182 ± 8 to 134 ± 8 ng-atoms of oxygen (ngatomO)/min/mg and induced a small but statistically significant increase in
from 143 ± 3 to 146 ± 3 mV. The shift of kinetic curves (the change in respiratory rates at the same value of
) indicated that 20 µM of 2,2`,5,5`-TCB affected the all three subsystems of oxidative phosphorylation: it increased the membrane leak of the inner membrane (Fig. 3A
) and inhibited the respiratory (Fig. 3B
) and phosphorylation subsystems (Fig. 3C
). Although the shift in kinetics of the membrane leak caused by 2,2`,5,5`-TCB was not large (Fig. 3A
), the flux through this subsystem JL in State 3 increased by more than twice (114%). In addition, 2,2`,5,5`-TCB inhibited the fluxes through the respiratory subsystem JR (by 26%; Fig. 3B
) and phosphorylation subsystem JP (by 32%; Fig. 3C
).
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Essentially the same pattern of the 2,2`,5,5`-TCBinduced changes was determined when modular kinetic analysis on mitochondria respiring with glutamate + malate was performed (Fig. 4). The rate of glutamate + malate oxidation (respiratory flux, JR) in State 3 was decreased by 2,2`,5,5`-TCB to a similar degree (23%) as in the case of succinate oxidation: from 141 ± 6 to 109 ± 7 ngatomO/min per mg. The respiratory (Fig. 4B
) and the phosphorylation (Fig. 4C
) subsystems were inhibited (the phosphorylation flux JP decreased from 134 ± 5 to 98 ± 7 ngatO/min per mg), but an increase in the membrane leak (Fig. 4A
) was smaller (JL in State 3 increased from 8 ± 1 to 11 ± 1 ngatO/min per mg) as compared with succinate oxidation. Despite the marked changes in the activities of the three subsystems induced by 2,2`,5,5`-TCB (Fig. 4
), the value of
in State 3 of mitochondria oxidizing glutamate + malate has remained the same (144 ± 2 in control and 143 ± 2 mV with 2,2`,5,5`-TCB), implying that the decrease in the activity of
-producing reactions (the respiratory subsystem) is completely compensated by a diminution in the activity of
-consumers (the membrane leak + the phosphorylation subsystem).
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DISCUSSION |
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In numerous mitochondrial studies, changes in separate functional parameters (e.g., rate of respiration in metabolic states 4 and 3, and/or uncoupled state, membrane potential, swelling) are measured with the aim of evaluating the influence of different effectors or damaging factors on oxidative phosphorylation. However, the established increases or decreases are only of minor informative value for understanding the molecular reasons of the observed changes. It appears that standard methods of enzymology are inadequate to deal with kinetics and control of intricate metabolic pathways. The metabolic control theory was developed (Kacser and Burns, 1973, 1979
; Heinrich and Rapoport, 1974
) for analyzing the kinetic behavior of multicomponent enzyme systems. The theory gives a qualitatively new point of view in understanding the metabolic control, because it considers not only properties related with individual processes, but also properties determined by inherent complexity of metabolic systems (systematic properties), reviewed in Fell (1997). Within the framework of this theory, a group of British researchers developed the "top-down" method (or modular kinetic analysis), which was first experimentally applied for the analysis of the fluxes in oxidative phosphorylation (Hafner et al., 1990
). This approach facilitates the analysis of a complex process by dividing it into a small number of modules, each comprising multiple enzymatic steps. By rather moderate experimental efforts (measuring the uptake of oxygen simultaneously with the membrane potential
and manipulating the fluxes with specific inhibitors), one may gain important information about the contribution of each subsystem to the changes induced by a multisite effector in the overall flux through the system. This method has been applied in different ways in different systems: isolated mitochondria, intact cells, perfused tissues, organs, and entire organisms (Ainscow and Brand, 1999
; Brown et al., 1990
; Borutaite et al., 1995
; Kavanagh et al., 2000
, Mildaziene et al., 1996
; Marcinkeviciute et al., 2000
). It has been successfully used for the study of toxic effect of cadmium ions on oxidative phosphorylation in potato mitochondria (Kesseler and Brand, 1994
).
In the present study we demonstrate how the modular kinetic analysis is used to identify the distribution of a multisite toxic effect of the organochemical pollutant 2,2`,5,5`-TCB on the oxidative phosphorylation system in rat liver mitochondria. This method allowed us to demonstrate that 2,2`,5,5`-TCB, even at low concentration, affects many sites in the machinery of mitochondrial energy transformation. It increases the membrane leak and inhibits both the respiratory and the phosphorylation subsystems, thus causing a slight (succinate oxidation) or substantial change (glutamate + malate oxidation) in . Because the resulting change in
reflects the balance of changes in the kinetics of
-producers and
-consumers, it may serve as a sensitive measure to evaluate the degree to which different blocks are affected. Interaction of 2,2`,5,5`-TCB with the myxothiazole-sensitive site in Complex III and succinate dehydrogenase (Nishihara et al., 1986
) may be deemed responsible for the inhibition of the respiratory subsystem in case of succinate oxidation. However, the analysis of
changes indicated stronger inhibition of the respiratory subsystem with NADH-dependent substrate (glutamate + malate) than with succinate as a respiratory substrate. This finding helped us to detect that Complex I in the respiratory chain was inhibited by 2,2`,5,5`-TCB. Although it was supposed in earlier studies (Nishihara et al., 1985
) that Complex I is not sensitive to 2,2`,5,5`-TCB, such assumption was not based on direct estimation of enzymatic activity. Contrary to this assumption, the results obtained by standard biochemical assay (Fig. 5
) clearly show that Complex I is inhibited by the toxin. Also, it may be possible that the binding of 2,2`,5,5`-TCB to components of oxidative phosphorylation depends on temperature, as succinate oxidation was inhibited to a much higher extent compared with glutamate + malate when the experiments were performed at 25°C (Nishihara and Utsumi, 1986
), whereas at a physiologically more relevant temperature (37°C), oxidation of the both substrates was inhibited by 20 µM 2,2`,5,5`-TCB to the same extent (Figs. 3 and 4
).
The observed effects of 2,2`,5,5`-TCB on the membrane leak and the respiratory chain are in full agreement with the results reported by other authors (Nishihara et al., 1985, Nishihara et al., 1986
). Although nonplanar TCBs were reported to show strong uncoupling action (Nishihara et al., 1985
), it is doubtful that TCB can act as a traditional uncoupler (the lipophilic proton carrier). The uncoupling of oxidative phosphorylation by TCBs could possibly be explained by changes in the membrane properties after incorporation of the unusual lipophilic compound into the membrane structure.
In this study we demonstrate the usefulness of modular kinetic analysis in the detection of toxic effects on the phosphorylation subsystem. These effects cannot be estimated in a direct way, because in mitochondria oxidizing different substrates, the flux of ATP synthesis (phosphorylation) is largely controlled by the other two subsystems, mostly by the respiratory subsystem (Hafner et al., 1990; Mildaziene et al., 1996
, 2000
). Therefore, any factor disturbing the activity of the respiratory subsystem (or the membrane leak) via induced changes in
elicits secondary effects on the phosphorylation flux. In modular kinetic analysis, the flux of phosphorylation is estimated by subtracting the portion of oxygen consumption determined by the membrane leak JL from the flux through the respiratory subsystem JR at the certain value of
(i.e., JP = JR JL). Thus, this approach provides a valuable tool for the detection of changes in JP kinetics per se induced by an effector. In mitochondria oxidizing both succinate and glutamate + malate, we obtained an obvious shift toward lower activity in the kinetics of the phosphorylation subsystem. A noticeable decrease in the oligomycin-binding sites indicated interaction of 2,2`,5,5`-TCB with ATP synthase, and inhibition of ATPase by 2,2`,5,5`-TCB was confirmed by the standard biochemical assay of the ATP hydrolytic activity. Therefore, we suppose that ATP synthase is at least one of the targets of 2,2`,5,5`-TCB determining inhibition of the phosphorylation subsystem and contributing significantly to the overall 2,2`,5,5`-TCB effect on oxidative phosphorylation.
We conclude that modular kinetic analysis is a useful tool for the evaluation of multisite effects of toxins, and here we present an example of how it could be used for the identification of targets of noxious action of 2,2`,5,5`-TCB on the oxidative phosphorylation system in rat liver mitochondria. This approach allowed us to decode the pleiotropic nature of the impairment of mitochondrial respiration by 2,2`,5,5`-TCB. We show that disturbance in the activity of all three subsystems (or modules) contributes to the overall inhibition of oxidative phosphorylation, and in further analysis we reveal several individual components within the system that are sensitive to the toxin. We conclude that this lipophilic organic pollutant, which is easily incorporated in the membrane, has a rather unspecific action as it affects the membrane leak, Complexes I, II (succinate dehydrogenase), and III (cytochrome bc1) in the respiratory chain and ATP synthase. We can only speculate on the extent to which these effects are caused by the change in composition and properties of the mitochondrial membrane and the lipid environment that is essential for function of the membrane proteins. However, at least for ATP synthase, a more specific interaction with 2,2`,5,5`-TCB may be supposed (as judged by the decrease in the oligomycin-binding sites). Whatever the molecular details of the 2,2`,5,5`-TCB interaction with components of oxidative phosphorylation, it becomes clear that by simultaneously acting on many steps, it efficiently inhibits the physiologically important fluxes of respiration and ATP synthesis in liver mitochondria. Most probably, other congeners of 2,2`,5,5`-TCB share this property to a great extent. Therefore, modular kinetic analysis will be helpful to elucidate the relationship between their toxic power and ability to interact with different components within the oxidative phosphorylation system.
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ACKNOWLEDGMENTS |
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NOTES |
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