A study on the mechanism of energy coupling in the redox chain

Generation of a membrane potential in the respiratory chain-deficient particles of beef heart mitochondria has been studied. For detection of membrane potential, phenyl dicarbaundecaborane (PCB^–,) and anilinonaphthalene sulphonate (ANS^–) probes were used. The respiratory chain-deficient submitochondrial particles were prepared after Arion and Racker (E-SMP), the procedure including complete disappearance of membrane structures and subsequent reconstitution of membrane vesicles as judged by the electron microscopy study. E-SMP were found to be deficient in cytochromesa,a ₃ and transhydrogenase, the cytochromeb,c ₁ andc content being lowered. Addition of NADH, succinate and tetramethyl-p-phenylenediamine+ascorbate did not induce either any oxygen consumption or membrane potential formation. Treatment of E-SMP with NADPH+NAD⁺ or with NADH+CoQ₀ did not entail generation of membrane potential, in contrast to that of parent, pyrophosphate submitochondrial particles (PP-SMP).E-SMP displayed an oligomycin-sensitive ATPase activity which could be increased by reconstitution of E-SMP with coupling factor F₁. Addition of ATP resulted in an uptake of PCB^– and enhancement of ANS^– fluorescence, the facts testifying to the formation of the membrane potential with plus inside E-SMP. Membrane potential formation was arrested by oligomycin, rutamycin, and uncouplers. Addition of respiratory chain inhibitors (antimycin+rotenone+ cyanide), complete reduction of respiratory carriers by dithionite and oxidation by ferricyanide were without effect on ATP-supported formation of membrane potential in E-SMP. It was concluded that utilization of ATP energy for the membrane potential generation does not depend on the state of the respiratory carriers and can be demonstrated under the conditions when none of redox chain coupling sites were functioning.Abbreviations PCB^– phenyl dicarbaundecaborane - ANS^– anilinonaphthalene sulfonate - E-SMP the respiratory chain-deficient submitochondrial particles - PP-SMP pyrophosphate submitochondrial particles     

Attenuation of sugar cataract by ethyl pyruvate

The effects of fluoxetine on the oxidative phosphorylation of mitochondria isolated from rat brain and on the kinetic properties of submitochondrial particle F₁F₀-ATPase were evaluated. The state 3 respiration rate supported by pyruvate + malate, succinate, or ascorbate + tetramethyl-p-phenylenediamine (TMPD) was substantially decreased by fluoxetine. The IC₅₀ for pyruvate + malate oxidation was 0.15 mM and the pattern of inhibition was the typical one of the electron-transport inhibitors, in that the drug inhibited both ADP- and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-stimulated respirations and the former inhibition was not released by the uncoupler. Fluoxetine also decreased the activity of submitochondrial particle F₁F₀-ATPase (IC₅₀ 0.08 mM) even though K_0.5 and activity of Triton X-100 solubilized enzyme were not changed substantially. As a consequence of these effects, fluoxetine decreased the rate of ATP synthesis and depressed the phosphorylation potential of mitochondria. Incubation of mitochondria or submitochondrial particles with fluoxetine under the conditions of respiration or F₁F₀-ATPase assays, respectively, caused a dose-dependent enhancement of 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. These results show that fluoxetine indirectly and nonspecifically affects electron transport and F₁F₀)-ATPase activity inhibiting oxidative phosphorylation in isolated rat brain mitochondria. They suggest, in addition, that these effects are mediated by the drug interference with the physical state of lipid bilayer of inner mitochondrial membrane.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Arrangement of the electric potential-generating redox chain in the mitochondrial membrane

The intramembrane arrangement of the respiratory chain generating electric potential difference across the mitochondrial membrane has been studied. The accessibility of various respiratory carriers to the non-penetrating electron donors and acceptors, such as ferri-and ferrocyanide, cytochrome c. fumarate and nicotinamide nuclcotides has been used as a test for surface localization of the carrier in the membrane of mitochondria and inside-out (sonicated) submitochondrial particles. Membrane potential formation was detected by measuring the transmembrane flows of the penetrating anion, phenyl dicarbaundecaborne (PCB^–).It is shown that ferricyanide reduction can support PCB^– movement if this electron acceptor interacts with intact mitochondria in the region localized on the oxygen site of the antimycin-sensitive point. The same region is accessible for ferrocyanide whose oxidation by O₂ can be also coupled with PCB^– translocation. Added nicotinamide nuclcotides cannot be utilized by mitochondria for supporting PCB^– movement.PCB^– movement in the inside-out submitochondrial particles can be supported by reduction of ferricyanide or fumarate by NADH, and of NAD⁺ by NADPH, the former process being sensitive to rotenone but not to antimycin. Antimycin-insensitive reduction of feericyanide or of CoQ₆ by succinate is not coupled with PCB^– transport. Neither ferrocyanide nor ferrocytochromec can be used as electron donors in the particles.Penetrating electron donors (TMPDH₂, succinate) and acceptors (menadione) are effective both in mitochondria and particles.It is coucluded that flavin and transhydrogenase regions of the potential-generating redox chain are localized near the inner surface, cytochromec region-near the outers surface of the internal membrane of intact mitochondria. It means that the redox chain includes at least one act of the transmembrane transfer of reducing equivalents between flavins and cytochromec.     

Specific inhibition by macrocyclic polyethers of mitochondrial electron transport at site I

The macrocyclic polyethers dibenzo-18-crown-6 (XXVIII) and dicyclohexyl-18-crown-6 (XXXI) inhibit the valinomycin-mediated K⁺ accumulation energized by glutamate, -ketoglutarate, malate plus pyruvate or isocitrate but not that promoted by succinate, ascorbate plus TMPD or ATP. The polyethers inhibit the oxidation of the former group of substrates without preventing either the oxidation of succinate or ascorbate plus TMPD or the hydrolysis of ATP.The substrate oxidation inhibited by the macrocyclic polyethers is relieved in intact mitochondria by increasing the concentration of K⁺ in the medium. It is also completely reverted by supplementing the medium with valinomycin, Cs⁺ and phosphate, or else by the addition of vitamin K₃.In submitochondrial sonic particles the macrocyclic polyethers inhibit the oxidation of NADH as well as the ATP-driven reversal of electron flow at the site I of the electron transport chain. They also block the oxidation of NADH in non-phosphorylating Keilin-Hartree particles as well as in Hatefi's NADH-coenzyme Q reductase. The polyethers do not inhibit electron transport in mitochondria from the yeast which lack the first coupling site.The inhibition of electron transport by the polyethers do not require of the addition of alkali metal cations such as K⁺ in intact mitochondria or other membrane preparations.It is established that the macrocyclic polyethers XXVIII and XXXI, already characterized as mobile carrier molecules for K⁺ in model lipid membranes, inhibit electron transport at site I of the electron transport chain from mitochondrial membranes.It is suggested that the ability of the polyethers to coordinate alkali metal cations in aqueous versus lipid environments, but not K⁺ transportper se, is related to their rotenone-like induced inhibition of electron flow in mitochondrial membranes.Supported in part by a Grant from the Research Corporation.     

The interaction of the potential-sensitive molecular probe merocyanine 540 with phosphorylating beef heart submitochondrial particles under equilibrium and time-resolved conditions

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 ( M540 ) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540 -membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 M NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membrane-binding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence of the electrolyte.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of potential-dependent light absorption changes of lipid bilayer membranes in the presence of cyanine and oxonol dyes

Summary The mechanism by which the light absorption of cyanine and oxonol dyes changes in response to changes in transmembrane electrical potential has been studied. Trains of membrane potential steps produce changes in the intensity of light passing through glycerylmonooleate (GMO) bilayer lipid membranes (BLM) in the presence of these dyes. The size of the signal-averaged absorbance change for one of the cyanine dyes diS-C₂-(5) is 10^–5. The response time for the absorbance change of all of the dyes was 10 sec. In order for an absorption signal to be observed, the concentration of dye on both sides of the membrane must be different. Since GMO bilayer membranes are permeable to the charged dyes that were studied, the dye concentration asymmetry necessary for the optical signal had to be maintained with a constant dc membrane potential, onto which the trains of potential steps were superimposed. The more hydrophobic dyes were the most permeant. Inclusion of cholesterol in the GMO bilayers decreased the permeance of the positively charged cyanine dyes, but increased the permeance of the negatively charged oxonol dyes. The magnitude and the size of the BLM absorbance change depended on the wavelength of illumination. Comparisons of the wavelength dependence of the BLM spectra with absorption difference spectra obtained with model membrane systems allow us to postulate a mechanism for a BLM absorbance change. For the cyanine and oxonol dyes, the data are consistent with an ON-OFF mechanism where a quantity of dye undergoes a rapid potential-dependent movement between a hydrocarbon-like binding site on the membrane and the aqueous salt solution near the membrane. For some dyes, which readily aggregate on the membrane, part of the absorbance change may possibly be explained by a potential dependent change in the state of aggregation of dye molecules localized on the membrane. Mechanisms involving a potential dependent change in the polarizability of the environment of membrane-localized dye molecules cannot be excluded, but seem unlikely.     

Energy-dependent accumulation of the uncoupler picrate and proton flux in submitochondrial particles

In the presence of ATP and oxidizable substrate, submitochondrial particles accumulate up to 7 nmol of picrate/mg of protein. Half of this value is reached at 5 μM picrate in the medium, and maximal energy-dependent accumulation occurs at 25 μM picrate. Mitochondrial proton fluxes calculated under such conditions are 0.80 and 1.08 pmol H⁺/cm²·sec at 10 μM and 25 μM picrate, respectively. These values are similar to those reported for state 4, and are therefore not large enough for uncoupling by picrate through proton translocation. The energy-dependent spectral response of oxonol VI is reversed to 50 % by 40 μM picrate, suggesting that abolishment of membrane potential is responsible for uncoupling of submitochondrial particles by picrate.     

Role of energy in oxidative phosphorylation

This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and P_i and for the release of ATP bound at the catalytic sites of F₁-ATPase. The process of ATP synthesis on the surface of F₁ from F₁-bound ADP and P_i appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a lowK _m (for ADP and P_i)-lowV _max mode and a highK _m -highV _max mode. TheK _m extremes for ADP are 2–3 µM and 120–150 µM, andV _max for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 s^–1 (mole F₁)^–1 at 30°C, which corresponds to 11 µmol ATP (min · mg of protein)^–1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunitc) of the membrane sector (F₀) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F₁, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F₁ in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential () and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions is exquisitely sensitive to changes in the rates of energy production and consumption.     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation     

A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.     

Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction

The effects of Tinopals (cationic benzoxazoles) AMS-GX and 5BM-GX on NADH-oxidase, NADH:ferricyanide reductase, and NADH APAD⁺ transhydrogenase reactions and energy-linked NAD⁺ reduction by succinate, catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in submitochondrial particles (SMP), were investigated. AMS-GX competes with NADH in NADH-oxidase and NADH:ferricyanide reductase reactions (K _i = 1 M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K _i = 5 M) mechanism. Neither compound affects reverse electron transfer from succinate to NAD⁺. The type of the Tinopals' effect on the NADH APAD⁺ transhydrogenase reaction, occurring with formation of a ternary complex, suggests the ordered binding of nucleotides by the enzyme during the reaction: AMS-GX and 5BM-GX inhibit this reaction uncompetitively just with respect to one of the substrates (APAD⁺ and NADH, correspondingly). The competition between 5BM-GX and APAD⁺ confirms that NADH is the first substrate bound by the enzyme. Direct and reverse electron transfer reactions demonstrate different specificity for NADH and NAD⁺ analogs: the nicotinamide part of the molecule is significant for reduced nucleotide binding. The data confirm the model suggesting that during NADH APAD⁺ reaction, occurring with ternary complex formation, reduced nucleotide interacts with the center participating in NADH oxidation, whereas oxidized nucleotide reacts with the center binding NAD⁺ in the reverse electron transfer reaction.     

Cell membrane water permeability of rabbit cortical collecting duct

Summary The water permeability (P _osm) of the cell membranes of isolated perfused rabbit cortical collecting ducts was measured by quantitative light microscopy. Water permeability of the basolateral membrane, corrected for surface area, was 66 m·sec^–1 for principal cells and 62.3 m·sec^–1 for intercalated cells. Apical membraneP _osm values corrected for surface area, were 19.2 and 25 m·sec^–1 for principal and intercalated cells, respectively, in the absence of antidiuretic hormone (ADH). Principal and intercalated cells both responded to ADH by increasingP _osm of their apical membranes to 92.2 and 86.2 ·sec^–1 respectively. The ratio of the total basolateral cell membrane osmotic water permeability to that of the apical cell membrane was 271 in the absence of ADH and 71 in the presence of the hormone for both cell types. This asymmetry in water permeability is most likely due to the fact that basolateral membrane surface area is at least 7 to 8 times greater than that of the apical membrane. Both cell types exhibited volume regulatory decrease when exposed to dilute serosal bathing solutions. Upon exposure to a hyperosmotic serosal bath (390 mosm), pricipal cells did not volume regulate while two physiologically distinct groups of intercalated cells were observed. One group of intercalated cells failed to volume regulate; the second group showed almost complete volume regulatory increase behavior.     

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

(1) Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-P_i exchange activity. (2) Aurovertin alone inhibits 50% of the oxonol shift at 2 μM, and no further inhibition occurs at up to 12 μM. In combination with even weakly effective uncouplers, 4 μM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. (3) No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10–27%, depending on the preparation. The extent of the oxonol response depends on the ATP / ADP ratio rather than the phosphorylation potential. (4) The dye response in the ATPase complex is 4–7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. (5) The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.     

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD⁺+NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD⁺+NADH both were added. The change was lacking in the presence of the F₀F₁-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F₀F₁-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K⁺-dependent F₀F₁-ATPase activity, suggesting a relationship between the F₀F₁-ATPase and K⁺ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.