WHAT REGULATES RESPIRATION IN MITOCHONDRIA?

Transcription

1 Vol. 39, No. 2, May 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages ] 9 WHAT REGULATES RESPIRATION IN MITOCHONDRIA? Bernard Korzeniewski Institute of Molecular Biology, Jagiellonian University, al. Mickiewicza 3, Krak6w, Poland Received March 4, 1996 Received after revision March 22, 1996 SUMMARY A (nearly) linear dependence of the on a given parameter value was proposed as the criterion for considering this parameter to be an efficient regulator of respiration in mitochondria. A number of possible candidates was tested using the dynamic model of oxidative phosphorylation developed previously, among others ADP, the ATP/ADP ratio, external phosphorylation potential and Atkinson's adenylate energy charge. Only the external phosphorylation potential Iog(ATP/ADP*Pi) was found to fulfil the above-mentioned criterion and thus it was proposed to be the parameter which 'actually' regulates oxidative phosphorylation in mitochondria. Key words: respiration, oxidative phosphorylation, regulation, liver mitochondria, ADP, ATP/ADP ratio, phosphorylation potential, adenylate energy charge. INTRODUCTION Mitochondria can respond to an increase in energy demand, by increasing the rates of respiration and ATP synthesis. In suspension of isolated mitochondria, for example, the addition of increasing amounts of hexokinase in the presence of glucose (an artificial ATP-usage system) leads to a subsequent increase in oxygen consumption, until mitochondria become saturated. This is what is called transition from state 4 (no hexokinase added) to state 3 (saturating amount of hexokinase). Different 'signals' have been proposed to regulate respiration and ATP synthesis in mitochondria in response to varying energy demand. Their list includes the concentration of ADP [1], ATP/ADP ratio [2,3], ATP/ADP*P i ratio [4], external phosphorylation potential Iog(ATP/ADP*Pi) [5,6] and adenylate energy charge (ATP+0.5*ADP)/(ATP+ADP+AMP) [7]. Nevertheless, there is still no general agreement as to what 'actually' regulates oxidative phosphorylation in mitochondria [8-10] /96/020415~05505.(30/0 Copyright 996 by Academic Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL One of the main reasons for such a situation seems to be that no clear criterion for the regulator searched has been formulated. Three external chemical compounds involved in energy metabolism are directly connected with the oxidative phosphorylation system in mitochondria, namely external ATP, ADP and inorganic phosphate. Two former are transported through the inner mitochondrial membrane by the ATP/ADP carrier and the latter is transported by the phosphate carrier. In a sense, the concentration of each of these metabolites influences the oxidation and phosphorylation fluxes in mitochondria, at least within a certain range of its value. The same can be said about every parameter being a combination (function) of the three mentioned concentrations, such as phosphorylation potential and adenylate energy charge mentioned above (the concentration of AMP is related to concentrations of ATP and ADP via the reaction catalyzed by adenylate kinase). Furthermore, each of these parameters is in some way related to the others: when one parameter changes, then (in most cases) the other parameters also change; thus it is difficult to say which of these changes is correlated 'most directly' with the change in the. Therefore, I would like to propose a simple criterion of being an 'efficient' regulator of respiration. This criterion is a (nearly) linear relationship between the and the value of P in the whole range of the oxygen consumption rate between state 4 and state 3. The main reason for this choice is that in the case of the linear dependence, a significant change in the rate of respiration always is accompanied by a significant change in P and inversely. Therefore, we can assume that there is always a clear casual link between these two values. This is not the case for any kind of a non-linear relationship. Let us consider three most typical cases. In the saturable non-linear dependence, the is essentially constant at higher (saturating) values of P. Therefore, within this range the increase in P does not regulate oxygen consumption. On the other hand, the 'regulatory power' (relative changes in respiration divided by relative changes in P) at lower values of P is extremely high. A reversed situation takes place in the case of an exponential relationship. There P regulates respiration (very) efficiently only at higher values of P. At lower values of P, the value of this parameter does not influence the significantly. Finally, in a sigmoidal kind of non-linear relationship, P seems to regulate respiration only in the central, 'sigmoidal' range of the value of P. It should be emphasized that the dependence of respiration on P should be as linear as possible only in the range of P having 'physiological' significance, for example between state 4 and state3. It should be also stressed that the criterion proposed is slightly subjective and has no simple mechanistic meaning. THEORETICAL PROCEDURES The dynamic model of oxidative phosphorylation in rat liver mitochondria incubated with succinate, developed previously and verified for a broad range of conditions and system properties [11-13], was used for testing the possible candidates for an efficient regulator. In this model, each reaction (group of reactions) and process, taken into account explicitly, was 416

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL described by an appropriate kinetic equation. The rates of changes in time of independent variables were expressed as a set of ordinary differential equations. This set was solved numerically, by means of a computer. All kinetic descriptions and parameter values was the same as in [13]. The simulations performed in the present work consisted in increasing the value of the rate constant of the ATP usage (corresponding to addition of increasing amounts of hexokinase) from zero to the value which caused essentially no further increase in the. Thus, this theoretical procedure mimicked state 4 --~ state 3 transition in isolated mitochondda. For each new value of the rate constant of the ATP usage, a new steady state was reached after some time. Then, the values of the and each tested 'regulatory' parameter were recorded. Five parameters were tested: the concentration of ADP, ATP/ADP ratio, ATP/ADP*P i ratio, phosphorylation potential Iog(ATP/ADP*Pi) and adenylate energy charge (ATP+0.5*ADP)/(ATP+ADP+AMP). The values of these parameters were plotted against the subsequent values of the between state 4 and state 3. THEORETICAL RESULTS AND DISCUSSION The calculated interdependence between external concentration of ADP and the during state 4 ~ state 3 transition is presented in Fig. 1A. The relationship is strongly non-linear (the half-saturating ADP concentration is about p.m, in accordance with experimental results for liver mitochondda). [ADP] increases much faster with respiration at moderate values of the (state 3.5) than at low oxygen consumption (near state 4). In the vicinity of state 3 (the highest rates of respiration) concentration of ADP begins even to decrease, which is caused by the shift in the equilibrium of adenylate kinase towards AMP production. Therefore, ATP cannot be considered as an 'efficient' regulator of respiration, at least in relation to the criterion proposed. Fig. 1B shows the dependence of the external ATP/ADP ratio on the. Again, the relationship is strongly non-linear. In most of the range of the value of this ratio, its change causes very small change in oxygen consumption. This is also the case in Fig. 1C, where the external ATP/ADP*P i ratio is plotted against the. None of the above ratios seems to be an appropriate regulatory factor, as their dependencies on the do not fulfil the requirement of linearity. Fig. 1D presents the simulated relationship between the external phosphorylation potential (proportional to Iog(ATP/ADP*Pi)) and the oxygen consumption flux during state 4 state 3 transition. This dependence closely approaches linearity. Such a near-linear flux-force relationship was also measured experimentally, in the frame of the Non-Equilibrium Thermodynamics (NET) [14,15]. According to the criterion formulated in this paper, the external phosphorylation potential seems to be an efficient regulator of the (as correlated linearly to this rate): a finite change in this potential causes a finite change in oxygen consumption in the whole range between state 4 and state 3. Finally, the dependence of the Atkinson's adenylate energy charge on the rate of respiration is presented in Fig. 1E. It can be seen that this energy charge is a very bad measure 417

4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL ~ 150 ~,100.< ,'m_ 13_ n n 0.02 t--.<. O.Ol i~- i... C i L I ~ -- '~'lff~ ~ ~ '~ I 7OO 600 o_ 500 O 400 ~" ~ ~" C <" ~" I < "~ _ ,,. i i i i i i 0" t-- o 0.80 c- >., c e 0.00 "O i 1 i i i i Fig. 1. Simulated relationship between concentration of ADP (p.m) (A), external [ATP]/[ADP] ratio (B), external [ATP]/[ADP][Pi] ratio (IxM-1) (C), external phosphorylation potential Iog([ATP]/[ADP][Pi] ) (D) or adenylate energy charge ([ATP]+0.5*[ADP])/([ATP]+[ADP]+[AMP]) (E) and the (arbitrary units) during state 4 --> state 3 transition. of the energy demand in the system. In a broad range between state 4 and state 3.5 (about 50 % of the state 3 respiration) there is essentially no change in the value of this parameter, while respiration increases 4 times. The conclusion must be drown, that 'something else' than the energy charge is the reason for this stimulation of oxygen consumption. The theoretical results obtained in the present work suggest that only the external phosphorylation potential fulfil the above formulated criterion of an 'efficient' or 'actual' regulatory factor of oxidative phosphorylation in rat liver mitochondria. All other parameters tested do not seem to be 'good' regulators. Especially, there is no unique dependence of the on the concentration of ADP. The above conclusion has only a phenomenological and not mechanistic meaning. However, if we treat mitochoodria as one metabolic unit, we can state that they are regulated rather thermodynamically than kinetically, at least while responding to a varying energy demand. 418

5 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL The regulation of oxidative phosphorylation by the external phosphorylation potential was proposed in the frame of the 'near equilibrium' hypothesis [5,6], which assumed that the respiratory chain was near thermodynamic equilibrium with this potential. However, the ATP/ADP carrier was essentially displaced from ei:luilibrium in the model used for simulations. Therefore, the efficient regulation of oxidative phosphorylation by Iog(ATP/ADP*Pi) does not imply the validity of the 'near-equilibrium' hypothesis. The theoretical results obtained in the present study are limited to liver mitochondria incubated with succinate, since the model used for simulations has not been sufficiently tested for other types of mitochondria and respiratory substrates. REFERENCES Chance, B. and Williams, G.R. (1955) J. Biol. Chem. 217, Akerboom, T.M., Bookelmann, H. and Tager, J.M. (1977) FEBS Lett. 74, Lemasters, J.J. and Sowers, A.E. (1979) J. Biol. Chem. 254, t. Erecir~ska, M., Stubbs, M., Miyata, Y., Ditre, C.M. and Wilson, D.F. (1977) Biochim. Biophys. Acta 462, Klingenberg, M. (1961) Biochemistry 2, Hassinen, J.E. (1986) Biochim. Biophys. Acta 853, Atkinson, D.E. (1968) Biochemistry 7, Brand, M.D. and Murphy, M.P. (1987) Biol. Rev. 62, Brown, G.C. (1992) Biochem. J. 284, Wilson, D.F. (1982) in Membranes and Transport (Martinosi, A.N., ed.) pp , Plenum Press, New York. Korzeniewski, B. and Froncisz, W. (1991) Biochim. Biophys. Acta 1060, Korzeniewski, B. and Froncisz, W. (1992) Biochim. Biophys. Acta 1102, Korzeniewski, B., (1996) Biophysical Chemistry 57, Rottenberg, H. (1979) Biochim. Biophys. Acta 549, Stucki, J.W. (1980) Eur. J. Biochem. 109,