JXB Advance Access originally published online on June 23, 2006
Journal of Experimental Botany 2006 57(10):2133-2141; doi:10.1093/jxb/erl006
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Opinion Paper |
Equilibration of adenylates in the mitochondrial intermembrane space maintains respiration and regulates cytosolic metabolism
1Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
2Department of Plant Physiology, Umeå Plant Science Centre, University of Umeå, 901 87 Umeå, Sweden
*To whom correspondence should be addressed. E-mail: igamberd{at}cc.umanitoba.ca
Received 24 March 2006; Accepted 29 March 2006
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Abstract |
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Adenylate kinase (AK) uses one each of Mg-complexed and free adenylates as substrates in both directions of its reaction. It is very active in the mitochondrial intermembrane space (IMS), but is absent from the mitochondrial matrix where low [ADP] upon intensive respiration limits the respiratory rate. AK activity in the IMS is linked to ATP/ADP exchange across the inner mitochondrial membrane by using ATP (imported from the matrix) and AMP as substrates, the latter provided by apyrase and other AMP-generating reactions. The ADP formed by AK is exported to the matrix (in exchange for ATP), providing a mechanism for regeneration of ADP during respiration. From the AK equilibrium, and taking pH values characteristic of subcellular compartments, [Mg2+] in the IMS is calculated as 0.4–0.5 mM and in the cytosol as 0.2–0.3 mM, whereas the MgATP:MgADP ratio in the IMS and cytosol is 6–9 and 10–15, respectively. These represent optimal conditions for transport of adenylates (via the maintenance of an ATPfree:ADPfree ratio close to 1) and mitochondrial respiratory rates (via the maintenance of submillimolar [ADPfree] in the IMS). This, in turn, has important consequences for mitochondrial and cytosolic metabolism, including regulation of the protein phosphorylation rate (via changes in the MgATP:AMPfree ratio) and allosteric regulation of mitochondrial and cytosolic enzymes. Metabolomic consequences are discussed in connection with the calculation of metabolic fluxes from subcompartmental distributions of total adenylates and Mg2+.
Key words: Adenylate kinase, apyrase, free magnesium, intermembrane space, metabolomics, mitochondrion, respiration
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Introduction |
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Knowledge of driving forces for metabolic fluxes is essential in rationalizing the increasingly complex datasets coming out from metabolomic research. In any living system, driving forces include redox control and energy balancing that are connected to the production and utilization of high energy-containing compounds (Weckwerth, 2003). In this respect, mitochondria are on the cross-roads of energetic metabolism in both plant and animal cells, although in photosynthetic tissues the main site of ATP production is the chloroplasts. In photosynthetic tissues, under light conditions, mitochondria are the site of oxidation of photorespiratory glycine, and they supply the cytosol with oxidized substrates, for example, 2-oxoglutarate, and with ATP to drive anabolic processes there, for example, sucrose synthesis (Gardeström et al., 2002).
The optimal operation of mitochondria is possible only at homeostasis of major different parameters limiting mitochondrial respiration. One of these parameters is the availability of ADP, which can be easily exhausted in the intermembrane space (IMS). Another important aspect concerns the ATPfree:ADPfree ratio in the IMS. In plants, the adenylate translocator operates at the ATPfree:ADPfree ratio close to 1 and is strongly inhibited by the opposite adenylate with a Ki of the same order as Km (Schünemann et al., 1993). The ratio depends directly on [Mg2+] which also strongly affects ATP synthase (Boyer, 2000), regulates activities of major enzymes in mitochondria and cytosol, and is likely to affect Ca2+ concentration in the IMS (Gilli et al., 1998; Malmendal et al., 1999).
Adenylate kinase (AK; EC 2.7.4.3 [EC] ) equilibrates the pools of free and Mg-bound adenylates in all organisms (Blair, 1970; Kleczkowski et al., 1990; Kleczkowski and Randall, 1991). There are several isozymes of AK in the cell, including one in the mitochondrial IMS, but not in the mitochondrial matrix (Igamberdiev and Kleczkowski, 2001). AK is usually more active than net metabolite conversions in plant cell compartments, including fluxes into and out of the adenylate pools. Equilibration of adenylates by AK has major consequences for the operation of enzymes and the regulation of metabolic pathways. AK establishes a link between the ratios of free and Mg-bound adenylates, the [Mg2+], and the inner membrane potentials of mitochondria and chloroplasts (Igamberdiev and Kleczkowski, 2001, 2003). Recently it was shown that AK activity is an intrinsic property of a major class of membrane transporters, where it optimizes the gating of energetically neutral or downhill substrate flows across membranes (Randak and Welsh, 2005).
Theoretical considerations that a powerful mechanism of equilibration of adenylates by AK is an important tool for the maintenance of respiratory homeostasis of plant mitochondria are presented here. Evidence is provided that mitochondria avoid ADP limitation (operate in state 3) and support a sustainable metabolic flux equilibrating adenylate ratios and Mg2+ in both IMS and the cytosol. Approaches to calculate major metabolic fluxes from empirically measured concentrations of adenylates, with consequences for plant metabolomics, are discussed.
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Conditions for AK operation in the IMS of mitochondria |
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In the IMS of mitochondria, AK activity is two to four times higher than the maximum rates of ATP synthase activity in the matrix (Roberts et al., 1997). That means that AK may completely equilibrate ATP, ADP, AMP, and subsequently Mg2+. Taking into account true substrates of AK (Khoo and Russell, 1979; Kleczkowski et al., 1990; Kleczkowski and Randall, 1991), the reaction can be presented as:
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 | (1a) |
ATP+AMP, and the K apparent (Kapp or below simply K) is defined as
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0.3 to 1.5) depending on [Mg2+] (Kleczkowski and Randall, 1991; Igamberdiev and Kleczkowski, 2001). The AK reaction (equation 1) is displaced to the right upon the addition of ADP to isolated mitochondria (Roberts et al., 1997). On the other hand, under in-vivo conditions, ADP from IMS is taken intensively by the adenylate translocator in exchange for ATP that is synthesized by ATP synthase in the mitochondrial matrix. This means that, in the IMS, the reaction in vivo could be displaced to the left, given an efficient supply of AMP. The concentration of AMP in the cytosol is relatively high (15–20% of total adenylates) (Stitt et al., 1982), and adenylates easily permeate through the outer mitochondrial membrane. The high cytosolic AMP concentration implies that there could be an additional mechanism for AMP production, possibly involving apyrase (see below), an enzyme producing AMP from ATP and/or ADP (Zancani et al., 2001).
Under conditions of ADP formation by AK (reverse reaction), MgADP and ADPfree are formed in equimolar amounts. [Mg2+] can be calculated in these conditions using the simplified formula:
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1/d (Igamberdiev and Kleczkowski, 2003). In non-hypoxic conditions, gradients of Mg2+ and free and Mg-bound adenylate concentrations exist between the IMS and cytosol. They can be calculated if the cytosolic ATPtotal:ADPtotal ratio and pH in the IMS and cytosol (see below) are known, assuming free exchange of adenylates through the outer mitochondrial membrane. Gradients of adenylate ratios between the cytosol and mitochondria have recently been proposed for legume nodules (Wei et al., 2004). |
Adenylate ratios upon AK equilibrium |
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MgATP:MgADP ratio
The MgATP:MgADP ratio is a driving force of processes where key enzymes use MgATP and release MgADP. For instance, this ratio (often incorrectly simplified to ATP:ADP) controls the rate of phosphoglycerate reduction and thus the flux via the Calvin cycle (Fridlyand et al., 1997). As ratios of free and Mg-bound adenylates are linked to [Mg2+] (Igamberdiev and Kleczkowski, 2001), [Mg2+] can be defined as a metabolism-driving potential that could be derived from ATP:ADP ratios as:
 | (2) |
ATPfree:ADPfree ratio
The [ATPfree]:[ADPfree] ratio is important for the operation of both mitochondrial and plastidial adenylate translocators since they exchange free adenylates only (Krämer, 1980). It can be derived, upon AK equilibrium, as:
 | (3) |
 | (4) |
 | (4a) |
As previously shown (Igamberdiev and Kleczkowski, 2003), gradients of free adenylate concentrations across the inner mitochondrial membrane reflect the values of membrane potential, which is the driving force for adenylate translocation. Since translocation of ATPfree is inhibited by ADPfree (and vice versa) in a micromolar range, changes of the ratio of free adenylates across the membrane represent a very sensitive mechanism for adenylate transport (Genchi et al., 1996). It also underlies the importance of the near-equilibrium reaction of AK (and creatine kinase in animals) for the dissipation of metabolite gradients of adenylates (Dzeja and Terzic, 2003). The metabolic channelling of ADP from AK to the translocator has been further emphasized by the demonstration of a direct coupling between AK and oxidative phosphorylation (Laterveer et al., 1996, 1997). This establishes adenylate equilibrium exactly at the site of translocation and favouring ATP/ADP exchange.
MgATP:AMPfree ratio
It has been suggested that the ATPtotal:AMPtotal ratio changes as a square of the ATPtotal:ADPtotal ratio and that even small changes of the latter lead to significant changes of ATPtotal:AMPtotal to activate/inhibit specific protein kinase activities (Hardie and Hawley, 2001). This conclusion has been made assuming a constant value for Kapp of AK. However, since only C, but not K, is constant (Blair, 1970; Kleczkowski et al., 1990; Kleczkowski and Randall, 1991), this conclusion needs to be revised. Also, in most, if not all, cases it is MgATP and AMPfree, but not total ATP and AMP, that are biologically active (Purich and Fromm, 1972). After dividing equation 1a by [AMPfree]2 and making simple transformations the following is obtained:
 | (5) |
For protein phosphorylation by AMPK as well as some other protein kinases, only MgATP is effective in the transfer of the phosphate group, while the allosteric site is specific to AMPfree species (Bronner et al., 2004). This is further evidence that it is the MgATP:AMPfree ratio, and not the ATPtotal:AMPtotal ratio, that is relevant for regulation. As an example, the cytosolic glutamine synthetase is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and protein phosphatases. The phosphorylation status of the glutamine synthetase changes during light/dark transitions depending in vitro on the ATP:AMP ratio (Finnemann and Schjoerring, 2000), which is, according to the above analysis, really the MgATP:AMPfree ratio. The opposite effects of growth regulators like gibberellic and abscisic acids are explained by their differential regulation of the AMP-activated protein kinase subfamily (Bradford et al., 2003).
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Interdependence of adenylate ratios at different pH values |
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A summary of effects of different ratios of adenylate species on metabolism, along with formulae for the calculation of [Mg2+] and adenylate ratios upon AK equilibrium, are presented in Table 1. These parameters define the basic conditions determining the rates of metabolic pathways and their allosteric regulation via availability of a given free or Mg-bound adenylate. On the other hand, stability constants for free and Mg-bound adenylates depend strongly upon pH (O'Sullivan and Smithers, 1979) and the knowledge about pH in a given compartment is essential to estimate [Mg2+] properly as well as concentrations of free and Mg-bound adenylates. In this respect, the pH value of 6.5 in the IMS in actively respiring tissues is possibly the lowest that can be considered (Moore and Rich, 1985), whereas in the cytosol it is above 7.0 (7.2–7.5 is a likely approximation) except under hypoxia (Pukar et al., 2004). Under AK equilibrium, [Mg2+] in the IMS can approach 0.5 mM, while in the cytosol it is close to 0.20–0.25 mM (Igamberdiev and Kleczkowski, 2003).
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Based on the formulae in Table 1 and pH-dependence of stability constants for adenylate species (O'Sullivan and Smithers, 1979), the dependence of ratios of free and Mg-bound adenylates on ATPtotal/ADPtotal were plotted at two pH values, one closer to the pH value in the IMS (6.5) and another to the pH value in the cytosol (7.5). Figure 1 shows that, under AK equilibrium, the MgATP:AMPfree ratio responds much more strongly to an increase in the ATPtotal:ADPtotal ratio at lower but not higher pH. Thus, even very small changes in total adenylate concentrations in the mitochondrial IMS (pH <7) will lead to drastic changes in the MgATP:AMPfree ratio, while in the cytosol (pH >7) the ratio should be more stable, tending to change more significantly when pH drops, for example, under hypoxia. This is also illustrated in Fig. 2, which shows that at a given ATPtotal:ADPtotal ratio or at ATPfree/ADPfree=1, the MgATP:AMPfree ratio is lowest at pH 7.2–7.5 and increases significantly upon pH change especially towards the acidic range. This means that significant changes in adenylate concentrations are more likely to trigger phosphorylation cascades, in particular in the cytosol. Indeed, in animal tissue, phosphorylation processes are abundant in the cytosol and this has important signalling consequences for the rest of the cell (Dzeja and Terzic, 2003; Almeida et al., 2004). For plants, phosphorylation processes in the cytosol, mediated by the AMP-activated kinase mechanism, have also been shown as the major signalling phenomena (Halford et al., 2004).
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How the adenylate equilibrium works: the AK/apyrase cycle supports mitochondrial respiration |
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 TopAbstractIntroductionConditions for AK operation...Adenylate ratios upon AK...Interdependence of adenylate... How the adenylate equilibrium... Consequences for metabolomic...ConclusionsReferences |
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The IMS of mitochondria is a site with a very high AK activity (Roberts et al., 1997). While in animal mitochondria the contribution of AK equilibrium to adenylate homeostasis is complicated by high activity of creatine kinase (Gellerich, 1992), in plant mitochondria creatine kinase is absent and adenylates are equilibrated exclusively by AK. Thus, it is possible to work out a computable scheme of adenylate levels in the plant mitochondria IMS, which is also linked to adenylate distribution in the cytosol. Figure 3 illustrates schematically how the AK equilibrium supports the continuous synthesis of ATP by maintaining homeostatic [ADPfree] in the mitochondrial IMS. In this scheme, the adenylate translocator in the inner mitochondrial membrane takes ADPfree from the IMS in exchange for ATPfree. The membrane potential makes this exchange possible due to the differences in charge of two adenylate species (ATP4– versus ADP3–). This leads to a depletion of ADP and an increase of ATP in the IMS. In leaves, the calculated value of the ATPfree:ADPfree ratio in the light in the mitochondrial IMS (assuming the ATPtotal:ADPtotal ratio is similar to the cytosolic due to a free exchange of adenylates through the outer membrane) is close to 1 which corresponds to [ADPtotal] of
0.4–0.5 mM in the IMS. In darkness, it will be even higher. This means that mitochondria operate in state 3 in vivo, i.e. avoiding ADP limitation. A constant supply of the substrate for oxidative phosphorylation (ADP) via AK equilibrium will maintain operative mitochondria and prevent side-effects due to accumulation of NADH and other reduced products of metabolism. The side-effects could include formation of the potentially harmful superoxide and other reactive oxygen species.
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The mechanism of AK-dependent ADP production in the IMS assumes a continuous AMP supply which, together with ATP imported across an inner mitochondrial membrane, fuels AK reaction in the direction of ADP synthesis (Fricaud et al., 1992; Gellerich, 1992; Zancani et al., 2001). Possible sources of AMP include reactions leading to the formation of various CoA-derivatives (Zeiher and Randall, 1991; Beuerle and Pichersky, 2002; Shockey et al., 2003), activation of amino acids for protein synthesis (amino-acyl tRNA synthetases) (Lavrik and Moor, 1984), or nucleotide pyrophosphatase (Moorhead et al., 2003). The latter breaks ATP down to AMP and pyrophosphate (PPi) independently of other metabolic fluxes, and the PPi can be further used as an alternative source of energy or split by inorganic pyrophosphatase. Yet another possible independent mechanism supplying AMP to support AK-driven ADP production in the IMS is the involvement of apyrase (Zancani et al., 2001).
Apyrase (ATP diphosphotransferase, E-type ATPase, NTPDase, EC 3.6.1.5 [EC] ) is the Mg-dependent enzyme that is ubiquitously and abundantly distributed in all tissues. Based on studies on animal and plant tissues, apyrases can be cytosolic, membrane-bound, and nuclear, and an association with the cytoskeleton was also demonstrated (Shibata et al., 2002). In animals, apyrase was also found in the IMS of mitochondria (Flores-Herrera et al., 1999). In plants, although this location has not been demonstrated directly, partially purified mitochondria contained an apyrase isozyme that was probably attached to the outer membrane (Zancani et al., 2001). This could provide a mechanism for AMP formation from ATP and ADP in the IMS and, subsequently, supporting respiration. Apyrase isozymes may differ in their specificity for ATP and ADP as substrates and, thus, have different metabolic roles (Kettlun et al., 2005). Down-regulation of expression of apyrase genes leads to phenotypes that are usually associated with malfunction of mitochondria, for example, male sterility of pollen (Steinebrunner et al., 2003). It should be noted that, even if apyrase is not present in the IMS of plant mitochondria, its high cytosolic activity and possible attachment to the outer mitochondrial membrane (Zancani et al., 2001) are likely to allow the AMP flux to feed the AK reaction. This has been shown clearly with the preparations of partially purified mitochondria (Zancani et al., 2001).
Figure 4 outlines the involvement of AK and apyrase in supporting respiration via a mechanism that dissipates energy accumulated by mitochondria. This ‘futile’ cycle may be necessary when other metabolic fluxes are limited or saturated to fuel the AK reaction, and generates non-limiting ADP concentration for sustainable mitochondrial respiration. The mechanism may be considered as analogous to the operation of non-coupled pathways of mitochondrial electron transport that allow mitochondrial respiration without ATP limitation; they operate in a futile manner, but prevent over-energization and over-reduction of the cell (Igamberdiev et al., 2001; Gardeström et al., 2002). In the AK/apyrase cycle (Fig. 4), the substrate of apyrase is MgADP rather than MgATP. This is based on assays of crude and partially purified activities, where higher activities of some apyrase isoforms with MgADP, as compared with MgATP, have been observed (Moustafa et al., 2003; Kettlun et al., 2005). A direct supply of ADP by apyrase via ATP hydrolysis is also possible, but high AK activity is likely to compete with this reaction, given comparable AMP and ADP concentrations in the cytosol (Stitt et al., 1982).
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Consequences for metabolomic research |
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Magnesium is a widespread cation in living systems with a total concentration frequently in the order of tens millimolar. However, a significant amount of Mg is always bound to different compounds, and [Mg2+] can vary significantly, frequently decreasing to levels strictly limiting activities of Mg-dependent enzymes (Ishijima et al., 2003). An actual experimental determination of [Mg2+] without displacing the equilibrium between [Mg2+] and Mg-complexed species is difficult, and an indirect approach, based on AK-governed equilibrium between Mg-free and Mg-bound adenylates, was proposed (Igamberdiev and Kleczkowski, 2001). This approach allowed for subcellular [Mg2+] to be calculated, in particular during dark–light and light–dark transitions when [Mg2+] can change dramatically in a few seconds.
In metabolomic research, it is relatively easy to determine total amounts of adenylates, i.e. ATP, ADP, and AMP. Adenylates are not distributed evenly in the cell, so it is important to obtain data on their subcellular amounts, especially in the cytosol, plastids, and mitochondria. Existing approaches are not perfect (e.g. membrane filtration of protoplasts) but with a certain approximation they are appropriate. From the total amounts of adenylates it is possible to get information on whether they comply with the AK equilibrium, i.e. if the values of [ATP][AMP]/([ADP])2 are within the range of the apparent AK constant (0.3–1.5); if they are, it is easy to calculate [Mg2+] (Igamberdiev and Kleczkowski, 2001). In this respect, reliable data were obtained for chloroplast stromata, the IMS of mitochondria and chloroplasts, and from the cytosol of green leaves; however, there is apparently no AK equilibrium in the cytosol of some heterotrophic tissues, for example, roots (Igamberdiev and Kleczkowski, 2001).
Since adenylates in the cytosol are equilibrated both by AK in the IMS and the cytosolic AK (Schlattner et al., 1994), there should be a gradient of [Mg2+] in the cytosol, with a higher concentration (up to 0.5 mM under intensive respiration) in the IMS and near mitochondria and chloroplasts, and a lower concentration (down to 0.2 mM) at some distance from these organelles. In this respect, it is notable to mention that, in the light, the pH of the cytosol increases, leading to an increased pH gradient between the cytosol and the IMS (Yin et al., 1993). This may have important consequences for the operation of cytosolic Mg-dependent and Mg-activated enzymes such as non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (Bustos and Iglesias, 2005) and for establishing higher cytosolic MgATP:MgADP ratios.
The ATPtotal:ADPtotal ratio in leaf cytosol in the light is 5–8 (Gardeström and Wigge 1988; Igamberdiev et al., 2001). As calculated from the equations in Table 1, the MgATP:MgADP ratio is 10–15. Under those conditions, the ATPfree:ADPfree ratio is 0.7–1 (calculation based on equation 3). These conditions are close to optimal for the adenylate translocator since it exchanges free adenylates only and is not inhibited by Mg-bound adenylates (Schünemann et al., 1993). High MgATP:MgADP ratios in the cytosol are important for driving metabolic fluxes, for example, glycolysis and sucrose synthesis. In darkness, the cytosolic ATPtotal:ADPtotal ratio is lower (2–3) (Gardeström and Wigge, 1988) corresponding to ATPfree <ADPfree. Thus, ATPfree export from mitochondrial matrix could be tightly coupled to ADPfree import on the same translocator.
An important consequence of adenylate equilibrium in the mitochondrial IMS is its effect on signalling networks, as outlined in Fig. 5. It has been shown that Mg2+ is an allosteric activator of Ca2+ binding to calmodulin (Gilli et al., 1998), with the latter activating target enzymes in response to a submicromolar increase in [Ca2+], which in turn is the result of changes in [Mg2+] in a millimolar range (Malmendal et al., 1999). When ATP levels decrease under hypoxic conditions and [Mg2+] increases, Ca2+ is released from mitochondria to the cytosol, where its concentration raises above 0.1 µM (Subbaiah et al., 1998). On the other hand, Mg2+ is a strong inhibitor of Ca2+ uptake into mitochondria (McCormack and Denton, 1986). Thus, the increase of [Mg2+] in the IMS of mitochondria under intensive respiration will lead to the accumulation of Ca2+ in the IMS and thus to the activation of Ca2+-dependent enzymes, including NADPH and NADH dehydrogenases in the inner mitochondrial membrane facing IMS (Møller and Rasmusson, 1998) and NAD-kinase in the IMS (Zielinski, 1998). Therefore, AK equilibrium affects metabolic events via the effects on NAD-kinase and on external and internal NADPH dehydrogenases and probably affects Ca2+ homeostasis, which represents a primary event in the signalling network (Fig. 5).
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The signalling network associated with adenylate equilibrium in the IMS of mitochondria has a direct effect on the operation of the tricarboxylic acid cycle. Upon the increase in reduction level of mitochondria, the isocitrate dehydrogenase reaction is inhibited and citrate is transported from mitochondria to the cytosol (Igamberdiev and Gardeström, 2003). In more detail, the mechanism involves inhibition of NAD-dependent isocitrate dehydrogenase and operation of the mitochondrial NADP-dependent isocitrate dehydrogenase in a reverse direction (Igamberdiev and Gardeström, 2003). This has important consequences for supplying reducing power (NADPH) for anabolic processes via reactions catalysed by cytosolic aconitase and NADP-isocitrate dehydrogenase and providing 2-oxoglutarate for amino acid biosynthesis (Fig. 5).
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Conclusions |
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The AK-mediated equilibration of adenylates in the IMS of mitochondria represents an important factor providing optimal conditions for metabolic fluxes in mitochondria and cytosol. By providing ADP to the ATP/ADP translocator in the inner mitochondrial membrane upon ADP depletion by mitochondral ATP synthase, the homoeostatic condition is maintained, supporting respiratory flux. The process, if run solely by the AK/apyrase cycle (Fig. 4), is based on the operation of ‘futile cycle’ dissipating a part of the energy produced by mitochondria. On the other hand, this cycle prevents limitation of respiration that could directly result in the formation of reactive oxygen species, imbalance of membrane potential, damage of membrane proteins and lipids, and other undesirable phenomena. Together with non-coupled pathways of mitochondrial electron transport (alternative oxidase and rotenone-resistant NADH and NADPH dehydrogenases) and with operation of uncoupling proteins, the AK-governed ADP supply in the IMS represents a powerful mechanism supporting mitochondrial respiration. This is particularly important for green plant tissues having a massive turnover of photosynthetically formed reducing power flowing out to mitochondria via interorganellar metabolic shuttles.
AK equilibrium, established in the IMS of mitochondria, has important consequences for the rest of the cell and, in particular, for the cytosol where it establishes the values and ratios of free and Mg-bound adenylates. Based on these ratios, it is possible to provide insights into the mechanisms supporting the operation of metabolic fluxes. The set of the ratios of free and Mg-bound adenylates together with [Mg2+], which can be derived from experimentally obtained subcellular data for total adenylates, is a basic parameter for calculating the energy state of the cell and for computing the rates of metabolic and signalling pathways.
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