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.     

Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli

The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of Escherichia coli. The protonmotive force, delta p, was determined by measuring the transmembrane chemical gradient of protons, delta pH, from the cellular accumulation of benzoate anions, and the electrical gradient, delta psi, from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+). The accumulation of lactose was also used to calculate the delta p in this lactose operon constitutive beta-galactosidase negative mutant. The phosphorylation potential, delta GP', was determined by measuring the cellular concentration of ATP, ADP, and inorganic phosphate. According to chemiosmotic principles, at steady state the phosphorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio delta p/delta GP' can be used to determine the H+/ATP ratio. Respiring E. coli cells, in mid-exponential phase of growth or incubated in buffer, at external pHs from 6.25 to 8.25 had a constant delta GP' of about 500 mV. The H+/ATP ratio was found to be 3 when the delta p value derived from lactose accumulation levels was used. However, when the delta p values derived from delta pH and delta psi were used in the calculations, the H+/ATP ratio varied from about 2.5 at external pH 6.25 to about 4 at pH 8.25. Arguments are presented for the hypothesis that the delta psi values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.     

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.     

Protonmotive force as the source of energy for adenosine 5''-triphosphate synthesis in Escherichia coli.

Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).     

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.     

The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles

The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated. Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential). Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.     

Adenosine 5''-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.     

A requirement for ATP for beta-galactoside transport by Bacillus alcalophilus.

Lactose-grown cells of Bacillus alcalophilus actively transported methylthio-beta, D-galactoside (TMG) in a range of pH values from 7.5 to 10.5 with a pH optimum at 8.5. The TMG was accumulated in a chemically unmodified form, and cell extracts failed to catalyze either ATP or P-enolpyruvate-dependent phosphorylation of TMG. At pH 8.5, the lactose-grown cells exhibited a transmembrane proton gradient (deltapH) of 1.38 units, interior acid, and a transmembrane electrical potential (delta psi) of -132 mV. Accordingly, the total protonmotive force at this pH was very low, -51mV. Several lines of evidence indicate that the protonmotive force or delta psi did not directly energize TMG transport but, rather, that ATP was directly required: (a) in cells treated with arsenate so that the delta psi was unaffected and cellular ATP levels were markedly lowered, TMG transport was inhibited in proportion to the reduction of cellular ATP, while electrogenic alpha-aminoisobutyric acid transport was not; (b) when a valinomycin-induced potassium diffusion potential was established in starved cells, alpha-aminoisobutyric acid transport, but not TMG transport, was stimulated; and (c) in a series of experiments in which the delta psi was rapidly abolished by treatment with gramicidin, ATP levels declined slowly and the rate of TMG transport correlated directly with ATP levels rather than with the delta psi. Consumption of cellular ATP concomitant with TMG transport could be demonstrated.     

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Features of apparent nonchemiosmotic energization of oxidative phosphorylation by alkaliphilic Bacillus firmus OF4.

Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A. A., and Krulwich, T. A. (1989) Annu. Rev. Microbiol. 43, 435-463). To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5. As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential. The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient. Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout. When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5. The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes. Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells. A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work. A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.     

L-malate transport and proton symport in vesicles prepared from Pseudomonas putida

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV. In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.     

The effect of beta-galactosides on the protonmotive force and growth of Escherichia coli

The effect of three beta-galactosides on the components membrane potential (delta psi) and pH gradient (delta pH) of protonmotive force and growth of Escherichia coli has been examined. A good correlation between the reduction of the protonmotive force and growth inhibition was observed. Thus some galactosides had little effect on either the protonmotive force or growth while lactose diminished the protonmotive force and caused growth inhibition. This effect of lactose was dependent on the ionic composition of the growth media. In Medium A (77 mM-Na+, 85 mM-K+) lactose diminished delta psi but had no effect on delta pH. Growth inhibition was transient at an external pH 6.0 but complete at pH 7.5. In medium KA (approximately 1 mM-Na+, 162 mM-K+) delta pH was diminished and delta psi was not affected and consequently growth inhibition was complete at pH 6.0. In medium NA (163 mM-Na+, 20 mM-K+) lactose had little effect on delta psi, delta pH or growth. These data support Skulachev's hypothesis of buffering of the protonmotive force by K+ and Na+ gradients.     

Isotope and thermal effects in chemiosmotic coupling to the membrane ATPase of Streptococcus

We measured rates of ATP synthesis by the proton-translocating ATPase of the motile Streptococcus strain V4051. Starved cells were energized artificially by exposing their membranes to a variable electrical potential difference (internal medium negative) and a fixed pH difference (internal medium alkaline). The initial rates of ATP synthesis increased exponentially with protonmotive force. The results were the same in D2O and H2O; there was no solvent isotope effect. At a fixed protonmotive force, the rates were strongly dependent on temperature, as expected for a reaction with a large enthalpy of activation. At a different protonmotive force, the rates varied with temperature in an identical fashion; there was no change in the enthalpy of activation. We conclude that protonation-deprotonation steps are not rate limiting and that the protons that cross the membrane drive ATP synthesis by mass action. The transmembrane electric field acts by changing the concentrations of the reactants, not by changing the configuration of the enzyme-substrate complex.     

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.     

Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.     

The sodium cycle. II. Na+-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na+ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na+-motive respiratory chain of V. alginolyticus. Na+ loaded cells incubated in a K+ or Li+ medium fail to synthesize ATP in response to lactate addition. The addition of Na+ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na+ level. Neither lactate oxidation nor delta psi generation coupled with this oxidation is increased by external Na+ in the Na+-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse delta pNa, i.e., [Na+]in greater than [Na+]out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K+-loaded cells incubated in a high Na+ medium, i.e., when delta pNa of the proper direction [( Na+]in less than [Na+]out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K+ medium (delta pNa is low) is decreased by CCCP even without monensin. Artificial formation of delta pNa by adding 0.25 M NaCl to the K+-loaded cells (Na+ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na+ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na+ instead of H+ as the coupling ion.