Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.     

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.     

Gene duplication as a means for altering H+/ATP ratios during the evolution of FoF1 ATPases and synthases

In the evolution of the FoF1 family of proton-translocating membrane complexes, two reversals in function appear to have occurred, first changing it from an ATPase to an ATP synthase and then back again to an ATPase. Here we suggest that with each change in function, the ratio of protons transported per ATP hydrolyzed or synthesized (H+/ATP) was altered in order for the complex to better adapt to its new role. We propose that this was accomplished by gene duplication with partial loss in the number of functional catalytic sites (to increase H+/ATP) or functional proton channels (to decrease H+/ATP). This method of changing the H+/ATP ratio preserved overall structural features of the complex essential to energy coupling.     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration.     

Effects of deuterium oxide on elementary steps in the ATPase reaction. Evidence for the similarity of key intermediates in contractile and transport ATPase.

The effects of D2O on the elementary steps in the contractile and transport ATPase [EC 3.6.1.3] reactions were studied, and the following results were obtained: 1. The rate of H-meromyosin ATPase in the steady state decreased in D2O to 60% of that in H2O. Deuterium oxide did not affect the size or rate of the initial burst of Pi liberation, i.e. the amount or rate of formation of the reactive myosin-phosphate-ADP complex, MADPP. Moreover, neither the rate of change in the fluorescence spectrum of H-meromyosin induced by ATP (the rate of formation of the second enzyme-ATP complex, M2ATP) nor the rate constant of decomposition of MADPP into M degrees + ADP + Pi was affected by D2O. However, the equilibrium constant of the step M2ATP in equilibrium MADPP decreased in D2O to about 1/2 the value in H2O. 2. In the case of the Na+-K+-dependent ATPase reactin, neither the rate constant of formation of the second enzyme-ATP complex, E2ATP, nor that of decomposition of a phosphorylated intermediate, EADP approximately P, was affected by D2O. However, the equilibrium constant of the step E2ATP in equilibrium EADP approximately P decreased in D2O to about 1/2.5-1/4 of the value in H2O. These results suggest a similarity between the modes of binding of phosphate in MADPP in the myosin ATPase reaction and in EADP approximatley P in the Na+-K+-dependent ATPase reaction.     

Two types of kinetic regulation of the activated ATPase in the chloroplast photophosphorylation system

The inhibition by light of chloroplast coupling factor ATPase is not due simply to competing photophosphorylation. This inhibition is only partially relieved by either an arsenate-pool trap for released phosphate, or a pyruvate kinase/phosphoenolpyruvate trap for ADP. Moreover, the amount of product return that does occur in the absence of trapping systems, ascertained by incorporation of 32Pi or [2-3H]ADP back into ATP during the hydrolysis reaction, is insufficient to account for the observed activity decrease. In intermediate pi:H2O oxygen exchange studies, the number of water oxygens incorporated into each molecule of Pi produced does not vary with light intensity during the ATPase assay. This indicates that the light-induced change in ATPase activity is not due to an alteration of rat constants involved in the forward and reverse partitioning of the E.ADP.Pi complex. In contrast, ammonium chloride, an uncoupler of photophosphorylation which stimulates membrane-bound coupling factor ATPase when added after light activation, causes a shift in the pattern of intermediate Pi:H2O oxygen exchange toward a lower number of water oxygens incorporated per Pi formed. The effect of NH4+ consistent with ATPase activity stimulation caused by enhanced partitioning forward of the E.products complex. These observations suggest the operation of two mechanisms of regulation of ATP ase activity during chloroplast de-energization. However, a direct effect of NH4+ on the coupling factor itself, independent of the membrane energization effect, cannot be ruled out by the present studies. Additional oxygen exchange experiments lead to the conclusion that the binding of ATP at a site catalyzing extensive ATP:H2O back exchange in the native chloroplast system ( Wimmer, M. J., and Rose, I. A. (1977) J. Biol. Chem. 252, 6769-6775) is different from the binding of ATP for net hydrolysis in the system activated for ATPase.     

H+ ATPase of chromaffin granules. Kinetics, regulation, and stoichiometry

The chromaffin granule ATPase mediates an inwardly directed transport of H+ against concentration gradients, thereby forming and maintaining an electrochemical transmembrane H+ gradient. The kinetics of this ATPase, its activity modulation by changes in electrochemical H+ gradients, and the stoichiometry between H+ transport and ATP hydrolysis were studied in intact bovine chromaffin granules, resealed chromaffin granule ghosts, and highly purified fragmented chromaffin granule membranes. In fragmented membranes the H+ ATPase has a KM for ATP of 69 microM, a maximum of activity at pH 7.3, and a Vmax of 111 nmol/min/mg of protein at 20 degrees C. Trimethyl tin inhibits the ATPase at much lower concentrations than dicyclohexylcarbodiimide, whereas oligomycin, reserpine, and other inhibitors were without effect. In intact chromaffin granules, the ATPase activity was stimulated up to 300% by collapsing the H+ transmembrane gradients. H+/ATP stoichiometry was measured in resealed chromaffin ghosts devoid of ATP and catecholamines under conditions where no net pH changes occur upon ATP hydrolysis. After addition of ATP, the rates of H+ accumulation in the ghosts and ATP hydrolysis were both linear for about 60-100 s, and the ratio of H+ to ATP was 1.71. These data indicate that the H+ ATPase of chromaffin granules has both kinetic similarities and dissimilarities with other known H+ ATPases. The regulation by changes in H+ gradients and the fixed H+/ATP ratio of this ATPase is further evidence of its primary role in establishing electrogenic H+ translocation and H+ gradients in chromaffin granules.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Quantitative analysis of some mechanisms affecting the yield of oxidative phosphorylation: Dependence upon both fluxes and forces

The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H⁺ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic properties, regulation, modification of intrinsic stoichiometry).The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation.(1) By manipulating the actual flux through each of the respiratory chain units at constant protonmotive force in yeast mitochondria, we show that the ATP/O ratio decreases when the flux increases. Moreover, the highest efficiency was obtained when the respiratory rate was low and almost entirely controlled by the electron supply. (2) By using almitrine in different kinds of mitochondria, we show that this drug leads to a decrease in ATP synthesis efficiency by increasing the H⁺/ATP stoichiometry of ATP synthase (Rigoulet M et al. Biochim Biophys Acta 1018: 91-97, 1990). Since this enzyme is reversible, it was possible to test the effect of this drug on the reverse reaction of the enzyme i.e. extrusion of protons catalyzed by ATP hydrolysis. Hence, we are able to prove that, in this case, the decrease in efficiency of oxidative phosphorylation is due to a change in the mechanistic stoichiometry of this proton pump. To our knowledge, this is the first example of a modification in oxidative phosphorylation yield by a change in mechanistic stoichiometry of one of the proton pumps involved. (3) In a model of polyunsaturated fatty acid deficiency in rat, it was found that non-ohmic proton leak was increased, while ohmic leak was unchanged. Moreover, an increase in redox slipping was also involved, leading to a complex picture. However, the respective role of these two mechanisms may be deduced from their intrinsic properties. For each steady state condition, the quantitative effect of these two mechanisms in the decrease of oxidative phosphorylation efficiency depends on the values of different fluxes or forces involved. (4) Finally the comparison of the thermokinetic data in view of the three dimensional-structure of some pumps (X-ray diffraction) also gives some information concerning the putative mechanism of coupling (i.e. redox loop or proton pump) and their kinetic control versus regulation of mitochondrial oxidative phosphorylation.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

The proton-translocating ATPase of Candida tropicalis plasma membrane

Proton translocation activity of Candida tropicalis plasma membrane ATPase has been demonstrated using a fluorescent delta pH probe (ACMA) and by direct pH measurements. Modifications in fluorescence intensity and H+ transport are highly specific for Mg2+ and ATP, and are sensitive to the well-known inhibitors of the plasma membrane ATPase, vanadate and DCCD. A H+/ATP ratio of 0.54 is found.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Measurement of mitochondrial ATPase activity in maize root tips by saturation transfer p nuclear magnetic resonance

We show that saturation transfer ³¹P nuclear magnetic resonance can be used to measure the activity of the mitochondrial ATPase of maize (Zea mays L. hybrid WW × Br38) root tips in vivo. Unidirectional rates of ATP synthesis were determined in the steady state (i.e. ATP and cytoplasmic orthophosphate constant) under various conditions. These measurements, and determinations of oxygen consumption, give a P/O ratio (measured in the living tissue) close to 3. In succinate-fed root tips the P/O ratio is approximately 2. Cyanide inhibits the rate of ATP synthesis by two-thirds (P/O ratio ~1), with an effective inhibitor constant of ~35 micromolars. We show that the alternative electron pathway cannot make ATP, and does not normally operate in this tissue. This method of studying plant mitochondrial metabolism avoids potential artifacts encountered in studies using isolated and purified mitochondria. The method also allows, for the first time, direct and simultaneous examination of the relationship between the rate of ATP synthesis and levels of metabolites such as ATP, and derived parameters such as phosphorylation potential.     

Effect of eosin Y on ATPase activity of actomyosine complex of the uterus smooth muscle

The effect of eosin Y (2,4,5,7 - tetrabromofluorescein; 0.1-100 microM) on ATPase activity smooth muscle actomyosine was studied. The inhibition coefficient i50 of ATPase activity with eosin Y was 0.74 +/- 0.07 microM. The inhibitor decreased V(max) of actomyosine ATPase for ATP, but no influence on affinity of actomyosine for ATP was observed. It is suggested that eosin Y inhibits actomyosine ATPase activity noncompetitively in respect of ATP.     

The gastric [H,K]ATPase:H+/ATP stoichiometry

An H+/ATP ratio of 2 for H+ transport was determined from initial rate measurements at pH 6.1 in a purified gastric microsomal fraction containing the [H,K]ATPase. This ratio was independent of external KCl, though the apparent K0.5 for ATP was increased from 10.78 +/- 0.51 (n = 3) to 64.6 +/- 11.9 (n = 3) microM ATP and from 5.13 +/- 0.64 (n = 3) to 65.2 +/- 0.64 (n = 3) microM ATP for H+ transport and the K+-stimulated ATPase, respectively, as K+external was increased from 12 to 150 mM. The H+/ATP ratio was also relatively independent of ATP concentration. Maximum initial rates obtained in KCl-equilibrated vesicles were independent of added valinomycin, though net H+ transport was increased 29.3 +/- 1.03% (n = 6) by the addition of ionophore. Maximum net H+ transport in this vesicle preparation was 185 +/- 2.1 (n = 14) nmol mg-1 of protein. Initial rate measurements of ATPase represent a burst of K+-dependent activity of approximately 10-15 s duration. The H+/ATP stoichiometry was calculated based on the K+-stimulated component of hydrolysis. Under most conditions, the Mg2+-dependent component of hydrolysis was less than 10% of the (Mg2+ + K+) component of hydrolysis.     

Cyclosporin a inhibits the protonophoric uncoupling activity of laurate in liver mitochondria

The effects of cyclosporin A, carboxyatractylate, and glutamate on the protonophoric uncoupling activity of laurate in liver mitochondria have been studied. It was found that 5 μM cyclosporin A partly inhibits laurate-stimulated mitochondrial respiration, which is suggestive of its recoupling effect, i.e., the ability to suppress the protonophoric activity of this fatty acid. Under these conditions, cyclosporin A has no effect on the ability of carboxyatractylate and glutamate to inhibit the uncoupling effect of laurate. In their turn, these compounds do not influence the recoupling activity of cyclosporin A. The recoupling effects of cyclosporin A, carboxyatractylate, and glutamate are additive: acting simultaneously, they fully suppress the uncoupling activity of laurate. It is concluded that the protonophoric uncoupling activity of fatty acids in liver mitochondria is mediated not only by ADP/ATP and aspartate/glutamate antiporters, but also by a system that is sensitive to cyclosporin A, but is not related with cyclophilin D.     

Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange

The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO3 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. Carbonyl cyanide m-chlorophenylhydrazone and NH4Cl (uncouplers of photophosphorylation) and phloridzin (an energy-transfer inhibitor) also lower the rate of ATP-H2O back-exchange; they too are found to act by decreasing the turnover of the ATP pool, not the extent of exchange per reacting ATP molecule. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form. The mode of NH4Cl inhibition of the ATP synthesis reaction, therefore, is not through a change in the partitioning of the E X ATP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-dependent affinity change of Na+-binding sites of V-ATPase

V-type Na(+)-ATPase of Enterococcus hirae binds about six (6 +/- 1) Na(+) ions/enzyme molecule with a high affinity (Murata, T., Igarashi, K., Kakinuma, Y., and Yamato, I. (2000) J. Biol. Chem. 275, 13415-13419). After the addition of 5 mm ATP, the binding capacity dropped to about 2 (1.8 +/- 0.3) Na(+) ions/enzyme molecule, returning to the initial value concomitant with the decrease of ATP hydrolysis rate. These findings suggest that the affinity of four of six Na(+)-binding sites of the enzyme changes (lowers) in enzyme reaction. The ATP analogs (adenosine 5'-O-(3-thiotriphosphate) or 5'-adenylylimido-diphosphate), ADP, or aluminum fluoride that is postulated to trap ATPases at their transition state did not inhibit the Na(+) binding capacity significantly. Therefore, the affinity decrease of Na(+)-binding sites was unlikely to be due to ATP binding alone or at the transition state of ATP hydrolysis. In the presence of 5 mm ATP, the ATPase showed strong negative cooperativity (n(H) = 0.16 +/- 0.03) for Na(+) stimulation of ATPase activity. The Hill coefficient (n(H)) increased to 1 in parallel to the decrease of ATP concentration in the reaction mixture. Thus, the ATP-dependent affinity change cooperatively occurs in continuous enzyme reaction.     

Uncoupling of oxidative phosphorylation. 2. Alternative mechanisms: intrinsic uncoupling or decoupling?

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), oleic acid, and chloroform is further investigated by measuring in the presence of a certain concentration of each type of uncoupler (i) the mitochondrial P/O and respiratory control ratios upon progressive inhibition of the redox pumps and (ii) delta mu H and the rate of either electron transfer or adenosine 5'-triphosphate (ATP) hydrolysis in static head upon progressive inhibition of either the redox or the adenosine triphosphatase (ATPase) proton pumps. Chloroform exhibits in all the experiments a behavior very different from that of FCCP and oleic acid. For example, upon addition of antimycin to chloroform-supplemented mitochondria, the respiratory control ratio remains unchanged and the P/O ratio slightly increases (in a certain range of inhibition) instead of decreasing as expected for an increased membrane conductance (and as indeed measured in the presence of either FCCP or oleic acid). From the kinetic model of chemiosmotic free energy coupling described by Pietrobon and Caplan [Pietrobon, D., & Caplan, S.R. (1986) Biochemistry 25, 7690-7696] all the results can be simulated by making the assumptions that (i) chloroform acts specifically at the level of the proton pumps and intrinsically uncouples electron transfer and ATP hydrolysis/synthesis from proton translocation and (ii) FCCP and oleic acid have a mixed behavior and act both as protonophores and as intrinsic uncouplers of the redox pumps (but not of the ATPases). The consistency of the results with the alternative hypothesis that the three agents interfere either with localized energy coupling sites or with a direct interaction between proton pumps is discussed.