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matrix ADP and ATP in mitochondria Print E-mail
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On the ADP and ATP in mitochondria matrix.

(extracted from the letter of Dr. Chinopoulos to MITOCHONDRIA news list, with insignificant modifications)

   “One should be ready to be confronted with a vast literature on matrix adenine nucleotide measurements with considerable uncertainties.”

Question: “What are the mitochondrial matrix free ADP concentrations in normal and in pathological conditions? What are the major causes for the change, if there is any?”

Dr. Christos Chinopoulos:

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   Free matrix ATP and ADP values depend mostly on mitochondrial membrane potential, phosphate concentration and mitochondrial pH gradient, as well as other parameters (33 in all), detailed in [15].
   Regarding matrix ADP under “normal and pathological conditions”. Lets first define what we consider “normal” and what is “pathological” conditions. We consider State III and State IV respiration conditions as “normal” and State IVu (uncoupler-induced) as “pathologic”. Our model [15] predicts that the concentrations of ADP and ATP in the matrix of mitochondria at State III (mitochondrial membrane potential is about -145 mV) are equal to 8.7 mM and 3.3 mM, respectively. A transition from State III to State IV (mitochondrial membrane potential is about -170 mV) reverses the order of the concentrations to 2.2 mM for ADP and 9.8 mM for ATP. We reiterate, matrix ADP and ATP concentrations strongly depend on the amplitude of the membrane potential. These membrane potential values are for isolated mitochondria.
   For in situ mitochondria, membrane potential is lower at about -135 mV (Note 1). In order to compare the predicted values to experimental data, we measured matrix ATP and ADP concentrations in the extracts of mitochondrial matrix by HPLC. Assuming 1 microliter of matrix volume for every mg mitochondrial protein, we estimated the following values: At 0 mV (no respiration substrates and in the presence of 10-6 M of an uncoupler SF6847), rat liver mitochondria have (in mM):

  • AMP: 3.64 +/- 0.34;
  • ADP: 8.23 +/- 0.65;
  • ATP: 0.51 +/- 0.05.

   At -170 mV (mitochondria were energized with glutamate 5 mM + malate 5 mM), rat liver mitochondria have (in mM):

  • AMP: 2.57 +/- 0.67;
  • ADP: 2.98 +/- 0.41 (predicted 2.2 mM);
  • ATP: 7.11 +/- 1.55 (predicted 9.8 mM).

   Regarding measuring matrix ATP and ADP values in State III: it requires adding of ADP to the mitochondrial suspension, followed by its conversion to ATP. That creates a technical challenge, since the volume of mitochondrial matrix is about 2,000 times less than the volume of the incubation medium (2 ml). Therefore, the amount of adenylates in the matrix is many fold lower than that being present in the extramitochondrial compartment. This problem can be resolved by centrifuging mitochondria – while they are still being phosphorylating added ADP - through a layer of silicon oil (polymerized siloxanes; e.g., AP 150 from “Sigma”). This excludes virtually all water-soluble extramitochondrial adenylates while preserving all the content of the mitochondrial matrix.
   In isolated rat liver mitochondria, reports indicate a wide range of intramatrix free ATP to ADP ratios in State 3, ranging from 0.01 to 4.5 [2-4;8;9;12;22;24;27-30] or even to 8-12 [31;32]. In mitochondria in situ or “in vivo” most investigators agree on the 1-3 range [19;23;25]. In studies where isolated mitochondria were not separated from the incubation medium by means of centrifuging through silicone oil or where no corrections was made for adenine nucleotides retained in intermembrane space, the reported matrix ATP/ADP ratios trend to the higher values (3-4.5, e.g. [21]).

   It is also possible that the results obtained after separation of intra-and extramitochondrial compartments are not relevant because of the time used for the separation process and possible inter-conversions of adenine nucleotides even in the presence of inhibitors [2;3;12;18]. Furthermore, a great part of the matrix adenine nucleotides is bound to proteins [13], a notion supported by the fact that rat liver mitochondria retain more than 50% of their total adenine nucleotide content after permeabilization by toluene [14]. Because of this potential binding of adenine nucleotides to intramitochondrial proteins [1;6;11;20] the relationship between the measured total ATP/ADP ratio to free intramitochondrial ATP/ADP ratio is difficult to predict. Previous data by Vignais show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space [27]. It is therefore inferred that there are separate intramitochondrial pools of adenine nucleotides, one near the Adenine nucleotide translocase (ANT) – ATPase complex, and another pool being located in the “bulk” of the matrix volume. The notion of matrix microcompartmentation of adenine nucleotides emanated from several laboratories [5;16;17;26;27], but is not yet accepted unequivocally in the field [7;10;12].

   Overall, if one wish to know the concentration of free ADP in the matrix, it is required to estimate the mitochondrial membrane potential, mitochondrial pH gradient, and the concentration of mitochondrial inorganic phosphate (Pi). These are the most important parameters; some other less important parameters are detailed in ref [15]. One should be ready to be confronted with a vast literature on matrix adenine nucleotide measurements with considerable uncertainties.

Note 1. This is an average value. The respiration and phosphorylation conditions for mitochondria in situ are most likely somewhere between State III and State IV.See the Abstract at the end of this page.

Referenced publications.

[1] P.D. Boyer, Toward an adequate scheme for the ATP synthase catalysis. Biochemistry (Mosc.) 66 (2001) 1058-1066.
[2] F. Brawand, G. Folly, P. Walter, Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria. Biochim.Biophys.Acta 590 (1980) 285-289.
[3] E.J. Davis, L. Lumeng, The effects of palmityl-coenzyme A and atractyloside on the steady-state intra- and extra-mitochondrial phosphorylation potentials generated during ADP-controlled respiration. FEBS Lett. 48 (1974) 250-252.
[4] E.J. Davis, L. Lumeng, D. Bottoms, On the relationships between the stoichiometry of oxidative phosphorylation and the phosphorylation potential of rat liver mitochondria as functions of respiratory state. FEBS Lett. 39 (1974) 9-12.
[5] H.C. Hamman, R.C. Haynes, Jr., Elevated intramitochondrial adenine nucleotides and mitochondrial function. Arch.Biochem.Biophys. 223 (1983) 85-94.
[6] D.A. Harris, J. Rosing, R.J. van de Stadt, E.C. Slater, Tight binding of adenine nucleotides to beef-heart mitochondrial ATPase. Biochim.Biophys.Acta 314 (1973) 149-153.
[7] K.J. Hartung, G. Bohme, W. Kunz, Involvement of intramitochondrial adenine nucleotides and inorganic phosphate in oxidative phosphorylation of extramitochondrially added adenosine-5'-diphosphate. Biomed.Biochim.Acta 42 (1983) 15-26.
[8] H.W. Heldt, Differences between the phosphorylation potentials of adenosine triphosphate inside and outside the mitochondria. Biochem.J. 116 (1970) 15P.
[9] H.W. Heldt, M. Klingenberg, M. Milovancev, Differences between the ATP-ADP ratios in the mitochondrial matrix and in the extramitochondrial space. Eur.J.Biochem. 30 (1972) 434-440.
[10] H.W. Heldt, E. Pfaff, Adenine nucleotide translocation in mitochondria. Quantitative evaluation of the correlation between the phosphorylation of endogenous and exogenous ADP in mitochondria. Eur.J.Biochem. 10 (1969) 494-500.
[11] J.M. Jault, W.S. Allison, Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP. J.Biol.Chem. 269 (1994) 319-325.
[12] G. Letko, U. Kuster, J. Duszynski, W. Kunz, Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate. Biochim.Biophys.Acta 593 (1980) 196-203.
[13] C.J. Lusty, Carbamoylphosphate synthetase I of rat-liver mitochondria. Purification, properties, and polypeptide molecular weight. Eur.J.Biochem. 85 (1978) 373-383.
[14] M.A. Matlib, W.A. Shannon, Jr., P.A. Srere, Measurement of matrix enzyme activity in isolated mitochondria made permeable with toluene. Arch.Biochem.Biophys. 178 (1977) 396-407.
[15] E. Metelkin, O. Demin, Z. Kovacs, C. Chinopoulos, Modeling of ATP-ADP steady-state exchange rate mediated by the adenine nucleotide translocase in isolated mitochondria. FEBS J. 276 (2009) 6942-6955.
[16] M.S. Murthy, S.V. Pande, Microcompartmentation of transported carnitine, acetylcarnitine and ADP occurs in the mitochondrial matrix. Implications for transport measurements and metabolism. Biochem.J. 230 (1985) 657-663.
[17] T.A. Out, E. Valeton, A. Kemp, Jr., Role of the intramitochondrial adenine nucleotides as intermediates in the uncoupler-induced hydrolysis of extramitochondrial ATP. Biochim.Biophys.Acta 440 (1976) 697-710.
[18] E. Pfaff, M. Klingenberg, Adenine nucleotide translocation of mitochondria. 1. Specificity and control. Eur.J.Biochem. 6 (1968) 66-79.
[19] W.D. Schwenke, S. Soboll, H.J. Seitz, H. Sies, Mitochondrial and cytosolic ATP/ADP ratios in rat liver in vivo. Biochem.J. 200 (1981) 405-408.
[20] A.E. Senior, S. Nadanaciva, J. Weber, Rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase. J.Exp.Biol. 203 (2000) 35-40.
[21] E. Shrago, M. Ball, H.S. Sul, N.Z. Baquer, P. McLean, Interrelationship in the regulation of pyruvate dehydrogenase and adenine-nucleotide translocase by palmitoyl-CoA in isolated mitochondria. Eur.J.Biochem. 75 (1977) 83-89.
[22] E.A. Siess, O.H. Wieland, Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells. Biochem.J. 156 (1976) 91-102.
[23] S. Soboll, T.P. Akerboom, W.D. Schwenke, R. Haase, H. Sies, Mitochondrial and cytosolic ATP/ADP ratios in isolated hepatocytes. A comparison of the digitonin method and the non-aqueous fractionation procedure. Biochem.J. 192 (1980) 951-954.
[24] S. Soboll, R. Scholz, H.W. Heldt, Subcellular metabolite concentrations. Dependence of mitochondrial and cytosolic ATP systems on the metabolic state of perfused rat liver. Eur.J.Biochem. 87 (1978) 377-390.
[25] S. Soboll, H.J. Seitz, H. Sies, B. Ziegler, R. Scholz, Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell. Biochem.J. 220 (1984) 371-376.
[26] P.V. Vignais, Molecular and physiological aspects of adenine nucleotide transport in mitochondria. Biochim.Biophys.Acta 456 (1976) 1-38.
[27] P.V. Vignais, P.M. Vignais, J. Doussiere, Functional relationship between the ADP/ATP-carrier and the F1-ATPase in mitochondria. Biochim.Biophys.Acta 376 (1975) 219-230.
[28] E.I. Walajtys, D.P. Gottesman, J.R. Williamson, Regulation of pyruvate dehydrogenase in rat liver mitochondria by phosphorylation-dephosphorylation. J.Biol.Chem. 249 (1974) 1857-1865.
[29] R.J. Wanders, G.M. Van Woerkom, R.F. Nooteboom, A.J. Meijer, J.M. Tager, Relationship between the rate of citrulline synthesis and bulk changes in the intramitochondrial ATP/ADP ratio in rat-liver mitochondria. Eur.J.Biochem. 113 (1981) 295-302.
[30] O.H. Wieland, R. Portenhauser, Regulation of pyruvate-dehydrogenase interconversion in rat-liver mitochondria as related to the phosphorylation state of intramitochondrial adenine nucleotides. Eur.J.Biochem. 45 (1974) 577-588.
[31] D.F. Wilson, M. Erecinska, V.L. Schramm, Evaluation of the relationship between the intra- and extramitochondrial [ATP]/[ADP] ratios using phosphoenolpyruvate carboxykinase. J.Biol.Chem. 258 (1983) 10464-10473.
[32] D.F. Wilson, D. Nelson, M. Erecinska, Binding of the intramitochondrial ADP and its relationship to adenine nucleotide translocation. FEBS Lett. 143 (1982) 228-232.

 

 

Note 1 Abstract (the manuscript was submitted to EBEC 2011):

Time Lapse Measurement of Mitochondrial Membrane Potential in Absolute Millivolts in Single Intact Cells

Akos A. Gerencser1, Christos Chinopoulos2, Matthew J. Birket1, Martin Jastroch1, Cathy Vitelli1, David G. Nicholls1, Martin D. Brand1

1Buck Institute for Research on Aging, Novato, California, CA; 2Semmelweis University, Budapest, Hungary;

   Assaying mitochondrial membrane potential (delta psim) in absolute millivolts in intact cells had been previously limited mostly to radioisotope distribution methods. Here we introduce a purely fluorescence based delta psim   assay to calculate time courses of delta psim in absolute millivolts in monolayer cell cultures. We built a biophysical model-based method to calibrate single-cell fluorescence of a bis-oxonol-type plasma membrane potential (delta psip) indicator and the delta psim–probe TMRM to potentials. The delta psip-dependent distribution of the probes is modeled by Eyring rate theory, which we have verified using fluorescence imaging combined with voltage clamp. delta psim is determined in millivolts by deconvoluting TMRM fluorescence in time taking in account the slow, delta psip-dependent redistribution and its Nernstian behavior. The resting delta psim is calculated from a complete step-depolarization of delta psim. The calibration accounts for volume ratios, high and low affinity bindings, activity coefficients, background fluorescence and optical dilution. All of the calibration parameters are back-calculated from fluorescence intensities or measured by confocal microscopic assays (validated by electron microscopy), allowing comparisons of potentials in cells with different properties. We show that delta psim in cultured rat cortical neurons is regulated between -120 and -154 mV as a concerted effect of increased ATP turnover and Ca2+-dependent metabolic activation.


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