From the Departamento de Bioquímica, Instituto Nacional de Cardiología, México, D.F. 14080, México
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ABSTRACT |
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The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 ± 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 ± 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.
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INTRODUCTION |
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The notion that the cytosolic concentration of free
Mg2+
([Mg2+]c)1
had a constant value around 1 mM under different conditions
has changed in recent years. By using permeant fluorescent dyes and nuclear magnetic resonance, tissue-dependent variations in
[Mg2+]c in the range 0.4 of 0.8 mM have been observed, in response to several hormones and
agonists. For instance, norepinephrine induced a net release of
cellular Mg2+ (1), while vasopressin induced
Mg2+ accumulation in isolated hepatocytes (2). Likewise,
increments of 50% in [Mg2+]c has been
determined, after stimulation with the muscarinic agonist carbachol,
and a 10% increase was observed, after addition of forskolin in rat
sublingual mucous acini (3). In acinar pancreatic cells, addition of
acetylcholine or cholecystokinin-octapeptide promoted a significant
diminution in [Mg2+]c (4).
Arginine-vasopressin and endothelin-1 induced an increment in
[Mg2+]c in muscle cells, probably through a
Ca2+-mediated mechanism (5). Depletion of inositol
1,4,5-trisphosphate-sensitive Ca2+ stores, induced by
-adrenergic agonists, activated the uptake of Mg2+ by
these organelles (6).
Extracellular ATP stimulated the release of 40% of cellular Mg2+ in ascites cells (7); it was proposed that cAMP promoted Mg2+ release through the activation of a plasma membrane Na+/Mg2+ antiporter (8). However, a report indicating that addition of cAMP also induced a net release of 20-25% of total Mg2+ in rat liver mitochondria (9) was not confirmed (10). In beef heart mitochondria, the transition from basal (state 4) to active (state 3) respiration led to a small, but significant elevation in the mitochondrial matrix free Mg2+ concentration ([Mg2+]m) from 0.5 mM to 0.6-0.7 mM. This increase in [Mg2+]m persisted during ATP synthesis, until added ADP was exhausted; at this time [Mg2+]m returned to basal levels. These variations in [Mg2+]m were inhibited by oligomycin (11). An elevation in [Mg2+]m from 44 µM to 1.69 mM also induced a stimulation in the rate of citrulline synthesis in rat liver mitochondria (12). Modulation of mitochondrial glutaminase by 0-2 mM Mg2+ has also been observed (13).
All of these reports describing an active movement of Mg2+ in cells and mitochondria of different tissues and in response to different agonists suggest that Mg2+ may play a role as a second messenger in the cell. In this work, we show that variations of external Mg2+, and hence in [Mg2+]m, can modulate the activities of the 2-OGDH and the ATP synthase and, in consequence, Mg2+ may affect the rate of oxidative phosphorylation in isolated rat heart mitochondria.
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MATERIALS AND METHODS |
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Rat heart mitochondria were isolated from male Wistar rats of 250-300-g weight according to a previously described method using the protease type XXVII (Nagarse) from Sigma (14).
Dye Loading-- Heart mitochondria were loaded with Mag-Fura-2 or BCECF (Molecular Probes) by incubating 30-40 mg of mitochondrial protein in 2 ml of a medium composed of 250 mM sucrose, 10 mM HEPES, 1 mM EGTA (SHE medium), 1 mM MgCl2, 1 mM ADP, 0.2% fatty acid-free bovine serum albumin, pH 7.4, and 5 µM Mag-Fura-2/AM or BCECF/AM at 25 °C for 20 min. At the end of this incubation period, mitochondria were diluted 10-15 times with ice-cold SHE medium + 0.2% bovine serum albumin, centrifuged, resuspended in 1 ml of SHE medium, and kept on ice until use. Mitochondria loaded by following this procedure showed higher respiratory control values than nonloaded mitochondria, 8.6 and 4.3 (n = 2), respectively, with 10 mM 2-oxoglutarate as a substrate.
Determination of [Mg2+]m-- Mag-Fura-2-loaded mitochondria (0.5 mg protein/ml) were incubated in 120 mM KCl, 20 mM MOPS, 0.5 mM EGTA (KME medium), 5 mM succinate, and 2 µM rotenone, at pH 7.25 and 30 °C. To avoid interference by matrix NAD(P)H fluorescence, 2-oxoglutarate was not used as an oxidizable substrate for determinations of [Mg2+]m. Fluorescence changes were monitored under smooth stirring and gassing with 100% O2 in an Aminco Bowman Series 2 spectrofluorometer. Excitation wavelengths were 340 and 398 nm and emission was collected at 483 nm. [Mg2+]m was determined from the fluorescence ratio signal (R, 398/340 nm). Rmax and Rmin were obtained at the end of each experiment. Rmin was generated by addition of 800 pmol of A23187/mg protein and sufficient EDTA-Tris, pH 8.0, to chelate all the Mg2+ present in the incubation medium; 0.005% (v/v) Triton X-100 was added to ensure complete Mg2+ equilibration across the membrane. Rmax was obtained after further addition of 70 mM MgCl2. Calculation of [Mg2+]m was made using the following equation (15):
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(Eq. 1) |
pH Determination-- BCECF-loaded mitochondria (0.5 mg of protein/ml) were incubated in KME medium containing 0.5 mM 2-oxoglutarate, 10 mM NaCl, 600 µM ADP, 3.5 µM oligomycin, 800 pmol of A23187/mg of protein and different concentrations of Mg2+, Pi, or acetate. For pH calculations, a calibration plot was generated incubating 0.5 mg of protein/ml in the medium mentioned above, at the desired pH, in the presence of 2 µM carbonyl cyanide m-chlorophenylhydrazone, 200 pmol of nigericin/mg of protein and 0.005% Triton X-100 to equilibrate all ion gradients. Excitation wavelengths were 450 and 500 nm; fluorescence was collected at 530 nm. The plot of pH values versus fluorescence ratio signal gives a straight line between pH 6.8 and 7.8.
ATP Synthesis-- Mitochondria (1 mg of protein/ml) were incubated in KME medium containing 0.5 mM 2-oxoglutarate or 5 mM succinate (+1 µM rotenone), 10 mM NaCl, 10 mM glucose, 30 units of hexokinase, and 5 mM 32Pi (specific activity, 1-1.5 × 106cpm/ml, Cerenkov radiation), at 30 °C. After 5 min, 1.2 mM ADP was added, and the reaction was stopped 30 s later by addition of 200 µl of 30% (w/v) cold trichloroacetic acid. Excess 32Pi was extracted as described previously using acetone + n-butyl acetate as organic solvents (16). Radioactivity of an aliquot of the aqueous phase was determined as 32Pi Cerenkov radiation in a scintillation counter.
Activity of 2-OGDH-- Mitochondria (1 mg of protein/ml) were suspended in KME medium containing 1 mM 2-oxoglutarate, 10 mM NaCl, 600 µM ADP, pH 7.25, and different concentrations of Mg2+ and Pi at 30 °C. Matrix NAD(P)H formed was determined following mitochondrial intrinsic fluorescence at 460 nm with the excitation wavelength at 340 nm. To obtain the fluorescence minimum, mitochondria were incubated in the absence of added substrates until endogenous substrates were depleted (approximately 5-8 min) (NAD(P)H = 0%); the fluorescence maximum was reached by adding 5 µM rotenone for complete reduction of NAD(P)+ (NAD(P)H = 100%) at the end of each experiment.
Membrane Potential ()--
Mitochondria (1 mg protein/ml)
were suspended in KME medium containing 0.5 mM
2-oxoglutarate, 10 mM NaCl, 5 µM safranin O, at 30 °C. Absorbance was recorded at 554
520 nm (17, 18), using a dual-beam SLM Aminco DW2000 spectrophotometer. Zero
was reached by addition of 1 µM carbonyl
cyanide m-chlorophenylhydrazone and 2 µM
rotenone at the end of each experiment.
Oximetry Assays-- Mitochondrial respiration was measured using an oxygen Clark-type electrode. Mitochondria (0.6 mg of protein/ml) were incubated in KME medium containing 1 mM 2-oxoglutarate, 10 mM NaCl, 1 or 5 mM Pi, and 800 pmol of A23187/mg of protein. After 5 min, 600 µM ADP was added, and the change in the rate of respiration was measured.
Matrix ATP and ADP Content-- Mitochondria (2.5 mg of protein/ml) were incubated in KME medium plus 5 mM succinate and 2 µM rotenone at 30 °C for 10 min under orbital shaking. Then, 3% (v/v) cold perchloric acid, 25 mM EDTA was added, the suspension was centrifuged, and the supernatant neutralized for enzymatic determination of ATP and ADP. Essentially identical results were obtained when mitochondria were previously sedimented in a microcentrifuge at 6-10 °C and further denaturalized by the addition of perchloric acid.
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RESULTS |
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The increase in the external Mg2+ concentration induced a proportional, but small elevation in [Mg2+]m in rat heart mitochondria (Fig. 1). This Mg2+ gradient ([Mg2+]m/[Mg2+]ex) showed a slope of 0.066, in the range 0-3 mM externally added Mg2+, indicating that Mg2+ does not easily equilibrate across the mitochondrial inner membrane, probably due to a slow Mg2+ influx, or to an active Mg2+ efflux. Similar results were previously reported for rat liver mitochondria (12). To accelerate the equilibration of Mg2+, the divalent cation ionophore A23187 was added. Fig. 1 shows that the ionophore modifies the steady-state concentration of matrix Mg2+, although equilibration with external Mg2+ was not complete. Since mitochondria incubated with A23187 conserved the H+ gradient, it was not unexpected that A23187 did not produce complete equilibration of Mg2+ concentrations across the mitochondrial membrane. Moreover, A23187 seems to be a weak Mg2+ ionophore due to a low affinity and poor mobility across the mitochondrial membrane (20-22). Nonetheless, the addition of A23187 allowed a more rapid manipulation of matrix Mg2+ in a lower range of external Mg2+ concentrations. The use of 1600 pmol of 4-bromo-A23187/mg of protein, instead of A23187, resulted in a curve very similar to that shown in Fig. 1 in the absence of ionophore (data not shown).
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The addition of A23187 to mitochondria incubated in the absence of added MgCl2 decreased [Mg2+]m from 0.49 to 0.02 mM and induced a significant diminution of matrix ATP/ADP ratio and the ADP + ATP content (see Fig. 1, inset). The further addition of 3 mM Mg2+, in the presence of A23187, increased [Mg2+]m from 0.02 to 1.15 mM and preserved matrix ATP/ADP ratio and ADP + ATP content at high values. Although a correlation between [Mg2+]m and the ATP/ADP ratio (or ATP content) was not found for mitochondria incubated with 3 mM Mg2+ and with or without A23187, it is apparent from the data of Fig. 1 that, in the presence of A23187, the addition of external Mg2+ modified both the [Mg2+]m and the ATP/ADP ratio, which may affect the rate of oxidative phosphorylation.
The rate of oxidative phosphorylation, assayed in the presence of A23187, depended on Mg2+ concentration in the incubation medium (Fig. 2). Since the sensitivity to Mg2+ depended on whether succinate (+rotenone) or 2-oxoglutarate (2-OG) was used (p < 0.05), the data of Fig. 2 suggest the existence of at least two sites of modulation by Mg2+. These sites may be located in the phosphorylating system (i.e. the ATP synthase or the adenine nucleotide translocase) during succinate oxidation, and at the level of 2-OGDH for 2-OG oxidation. Replacement of Mg2+ by Mn2+ also induced an activation of ATP synthesis, but at higher concentrations (K0.5 values for Mn2+ were 0.60 ± 0.047 mM (n = 3) with succinate and 0.92 ± 0.052 mM (n = 3) with 2-OG as a substrate).
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Since succinyl-CoA synthase also requires Mg2+, its contribution to the uptake of 32Pi was assayed. In the inset of Fig. 2, it is shown that substrate level phosphorylation by the Krebs cycle accounted for up to 40-50% of total ATP synthesis during oxidative phosphorylation with 2-OG as an oxidizable substrate. As substrate-level phosphorylation and oxidative phosphorylation with 2-OG showed different sensitivities to Mg2+, an effect of Mg2+ on sites different from succinyl-CoA synthase seemed likely.
To discard the participation of contaminating ATPases, in the ATP synthesis assays, hexokinase + glucose was used to capture ATP generated by oxidative phosphorylation. This prompted us to determine the Mg2+ dependence of hexokinase. Under the conditions of ATP synthesis (see Fig. 2), K0.5 values of hexokinase for ATP-Mg were 92 µM, in the presence of 400 µM ATP, and 31 µM in the presence of 100 µM ATP. These two concentrations of added ATP represent the maximal level of ATP synthesis (for 1 mg of protein/ml in 30 s at 30 °C) during oxidative phosphorylation with succinate and 2-OG, respectively. The sensitivity of hexokinase to Mg2+ revealed that this enzyme is not involved in the lower sensitivity of oxidative phosphorylation to Mg2+ with 2-OG as a substrate (see Fig. 2). However, in the presence of succinate, the sensitivity of oxidative phosphorylation to Mg2+ might result from a mixed response of both hexokinase and ATP synthase to Mg2+. However, an essentially identical sensitivity of oxidative phosphorylation to Mg2+ was observed in the absence of hexokinase (K0.5 = 0.13 ± 0.014 mM, n = 3), with succinate (+rotenone).
The change in the magnitude of the membrane potential, as estimated
from the distribution of TPP+, was initially used to
monitor indirectly variations in 2-OGDH activity in mitochondria
incubated with limiting concentrations of 2-OG (Fig.
3A). A membrane potential
() of 130 mV in the absence of added ADP and Mg2+ and
in the presence of A23187 and Pi, was determined. This
value increased to 140 mV by the increase in
[Mg2+]ex (Fig. 3A,
circles). With 600 µM ADP, steady state
diminished to 112 mV by increasing [Mg2+]ex
(Fig. 3A, squares), due to stimulation of ATP
synthesis. Although under these last conditions the oxidative system
was activated by Mg2+, the diminution in
indicated
that, at Mg2+ concentrations of 0-1.5 mM,
activation of the phosphorylating system of the pathway by
Mg2+ was predominant. At higher Mg2+
concentrations (>1.5 mM), activation of the oxidative
system prevailed over that of the phosphorylating system, resulting in
values larger than those obtained at zero Mg2+ (data
not shown).
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The enhancement of up to 162 mV by increasing
[Mg2+]ex, in the presence of ADP + Pi + oligomycin (Fig. 3A, triangles),
which was larger than that reached at the same concentration of
[Mg2+]ex in the absence of ADP, indicated
that ADP was a modulator of the Mg2+ activation. In the
absence of added Mg2+, removal of Pi markedly
diminished
(Fig. 3A, diamonds). In the
absence of Pi,
increased when
[Mg2+]ex was increased, but only to 105 mV.
This latter observation prompted us to determine the effect of
different concentrations of Pi on the activity of
2-OGDH.
In comparison to the [3H]TPP+ method, the
absorbance difference of safranin O (Fig. 3B) allows for
continuous monitoring of , and a large number of experiments with
the same mitochondrial preparation. Using the safranin O signal, the
increase in Pi concentration (in the presence of ADP + oligomycin) potentiated the activating effect of Mg2+ on
the steady state value of
(Fig. 3B). Thus, the
half-maximal stimulation of
by Mg2+ was decreased
and the maximal value of
was elevated by increasing Pi concentrations. This effect of Pi was not
apparent in the absence of added ADP. Similar results to those of Fig.
3B were obtained by measuring the
[3H]TPP+ distribution under the same
conditions (data not shown).
The activity of 2-OGDH was also measured, following the level of reduction of matrix pyridine nucleotides. In the absence of added Pi, the increase in [Mg2+]ex did not promote the generation of NAD(P)H (Fig. 4A). However, an increase in Pi concentration induced both a decrease in the K0.5 value for Mg2+ and an increase in the level of NAD(P)H reduction. The rate of respiration measured in the presence of ADP + oligomycin (state 4) was also stimulated by increasing [Mg2+]ex (Fig. 4B). Again, the presence of increasing Pi concentrations potentiated the stimulation by Mg2+, through a diminution in the K0.5 value for Mg2+ and an increase in the maximal rate of respiration. Thus, the data of Figs. 3 and 4 indicate that Pi, in the presence of ADP, potentiates the activating effect of Mg2+ on 2-OGDH activity.
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The study of the effect of different Pi concentrations on the Mg2+ sensitivity of ATP synthesis and state 3 respiration supported by succinate (+rotenone) revealed a negligible effect on the K0.5 value for Mg2+, indicating that the effect of Pi was exerted only at the Krebs cycle level. Lack of Mg2+ activation on state 4 respiration with succinate (+rotenone) as substrate and oligomycin (data not shown), discarded the possibility that Mg2+ activated the respiratory chain.
Matrix acidification brought about by the Pi uptake might be involved in Mg2+ activation of 2-OGDH. The activating effect of 5 mM Pi on the stimulation of matrix NAD(P)H formation by Mg2+ was not reproduced by addition of 10 or 20 mM acetate (data not shown); the final steady-state pH values in BCECF-loaded mitochondria incubated with 10 mM acetate or 5 mM Pi, in the presence of ADP, oligomycin, and A23187, were 6.91 and 6.88 with no added Mg2+, and 7.11 and 7.16 with 1 mM Mg2+, respectively. These results indicate that matrix acidification is not the mechanism involved in the Pi potentiating effect.
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DISCUSSION |
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The different sensitivity to Mg2+ of the rate of
oxidative phosphorylation, with 2-OG or succinate, suggested that
different sites in the pathway are involved in the interaction with
Mg2+. With the former substrate, the data on , matrix
NAD(P)H, and respiratory rates indicated that the main site of
interaction with Mg2+ was 2-OGDH.
Since ADP-Mg is the true substrate for the ATP synthase (24), increasing [Mg2+]m is expected to activate this enzyme. However, the substrates for adenine nucleotide translocase are free ADP and ATP (25). During ATP synthesis, external ADP exchanges with matrix ATP. As ATP has a higher affinity for Mg2+ than ADP, increasing [Mg2+]m is expected to inhibit the adenine nucleotide translocase activity through the diminution of the internal substrate. Therefore, the effect of increasing [Mg2+]m on the rate of oxidative phosphorylation is not readily apparent. As the elevation of external Mg2+, and hence [Mg2+]m, resulted in higher rates of ATP synthesis with succinate as substrate, it can be assumed that Mg2+ activation of the ATP synthase prevailed over Mg2+ inhibition of the adenine nucleotide translocase. Stimulation of the rate of oxidative arsenylation, an analogous process to oxidative phosphorylation, but without the participation of adenine nucleotide translocase (16), by Mg2+ using succinate (data not shown), supported the interpretation of an activating effect of Mg2+ on the ATP synthase.
In addition to a direct interaction of Mg2+ with the oxidative phosphorylation enzymes, Mg2+ might also perturb matrix Ca2+ homeostasis, and hence, affect the rate of ATP synthesis (16, 19, 26) (reviewed in Moreno-Sánchez and Torres-Márquez (27)). For instance, Mg2+ might compete with Ca2+ for the same binding sites in 2-OGDH. A decreased Ca2+ sensitivity by increasing Mg2+ has been observed for the NAD+-isocitrate dehydrogenase (28), whereas an enhanced Ca2+ sensitivity was described for the pyruvate dehydrogenase phosphatase (29). Although the sensitivity of 2-OGDH to Ca2+, at different Mg2+ concentrations, has not yet been determined, Panov and Scarpa (30) reported that 2-OGDH can be activated synergistically by both Mg2+ and Ca2+, implying the existence of different binding sites.
Panov and Scarpa (30) also determined a dissociation constant (Kd) for Mg2+ of 25 µM in the isolated 2-OGDH, with saturating concentrations of thiamine pyrophosphate, coenzyme A, and NAD+. Although such a Kd value for Mg2+ is lower than the K0.5 value obtained in this study (0.48 mM, see Fig. 2), it can be argued that the matrix concentrations of the 2-OGDH coenzymes in intact heart mitochondria may be limiting, and that 2-OGDH activity is not the only controlling step of the pathway (25). The value of the Kd or K0.5 for Mg2+ may establish the physiological relevance of variations in [Mg2+]m. Thus, a K0.5 value of 0.48 mM would appear as more physiologically relevant for modulating 2-OGDH activity and the rate of oxidative phosphorylation, since this concentration is in the range of [Mg2+]m in intact mitochondria (31, 32).
It should be noted, however, that the estimated K0.5 values for Mg2+ refers to the external Mg2+ concentrations, which were not fully equilibrated with the mitochondrial matrix by A23187 (cf. Fig. 1). Thus, the K0.5 value of 0.48 mM for external Mg2+ corresponds to a [Mg2+]m of 140 µM, which is slightly below the physiological range. Higher K0.5 values for Mg2+ were determined at low Pi concentrations. For instance, a K0.5 value of 1 mM for Mg2+ was observed in NAD(P)H formation with 1 mM Pi (see Fig. 4A). Such a K0.5 value was diminished to 0.5 mM by increasing Pi concentration up to 3 mM Pi. The corresponding [Mg2+]m for 1 mM external Mg2+ would be 350 µM, a value well within the physiological range. A variation in the cytosolic Pi concentration from 0.83 to 3.1 mM induced by epinephrine was established in rat heart (33). Therefore, physiological modulation of the 2-OGDH activity by Mg2+ may depend on the level of cytosolic (and matrix) Pi.
Other possible sites of modulation by Mg2+ during oxidative phosphorylation supported by 2-OG oxidation were the succinyl-CoA synthase, the ATP synthase and hexokinase (in the experiments of 32Pi incorporation into ATP). However, the Mg2+ sensitivity of these three enzymes showed that their saturation by Mg2+ was fully achieved at concentrations (<0.2-0.3 mM) that stimulated oxidative phosphorylation by less than 40%. The lack of stimulation of state 4 respiration by Mg2+ in mitochondria that oxidized succinate, in the presence of oligomycin, discarded an effect of Mg2+ at the level of the respiratory chain. Thus, these results indicate that 2-OGDH is one (but not the only) of the main controlling steps of oxidative phosphorylation (see also Moreno-Sánchez et al. (26)), at nonsaturing Mg2+ concentrations. In this respect, control of the rate of oxidative phosphorylation by changes in the spermine/Mg2+ rates, without a concomitant increase in [Ca2+]m, has been shown in dog pancreas mitochondria (19).
Modulation of the 2-OGDH activity by adenine nucleotides is well established (23). A synergistic effect by Ca2+ and adenine nucleotides has been described (28). Mg2+ also activates 2-OGDH (30) (this work), but in contrast to other enzyme effectors, the mechanism of action is by enhancing the catalytic enzyme capacity (kcat), rather than by increasing substrate affinity. Potentiation of the modulating effect of Mg2+ by Pi, although clearly demonstrated in this work, is somewhat puzzling. There is a report describing an activation of purified 2-OGDH by a high concentration of Pi (>10 mM), through the diminution of the K0.5 for 2-OG (34). Moreover, the Pi potentiating effect could be through promoting changes in matrix pH, since modulation of 2-OGDH activity by pH has also been reported (35). However, substitution of acetate for Pi, to induce similar matrix pH values, did not restitute the Mg2+ sensitivity of 2-OGDH. Thus, a direct interaction of Pi with the enzyme is likely to occur. From the present findings, the question that arises is to what extent and how Pi and Mg2+ affect the interplay of the other well described effectors, such as Ca2+ and adenine nucleotides, and the coenzymes NAD+, thiamine pyrophosphate, and coenzyme A, on 2-OGDH activity.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Gómez-Puyou and G. MacCarthy for their critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by CONACyT 2169-M9304, CONACyT F-554, PADEP/UNAM-05354, and PADEP/UNAM-05345 grants.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. de
Bioquímica, Instituto Nacional de Cardiología, Juan
Badiano #1, Col. Sección XVI, México, D.F., 14080, México. Fax: 525-573-0926.
1 The abbreviations use are: [Mg2+]c, cytosolic free Mg2+ concentration; [Mg2+]m, matrix free Mg2+ concentration; [Mg2+]ex, external Mg2+ concentration; 2-OGDH, oxoglutarate dehydrogenase; 2-OG, 2-oxoglutarate; TPP+, tetraphenylphosphonium; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; BCECF/AM, BCECF/acetoxymethyl ester; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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