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.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.     

Comparison of the energetics of lactose active transport: artificial versus enzyme-associated energy source

To further consider the thermochemical method as a useful approach for active transport research and to investigate the characteristic of a proton electrochemical potential (delta mu H+) across the membrane, the energetics of lactose active transport across Escherichia coli membrane vesicles coupled with an artificial electron donor (phenazine methosulfate-ascorbate) has been investigated. The results were compared with those obtained with an enzyme-associated electron donor (lactate dehydrogenase-D-lactate). The oxidation of an electron donor provided the energy necessary for the transport process. The observed higher heat of ascorbate oxidation reaction in the presence of a proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) further confirmed the formation of delta mu H+ across the membrane. Part of the oxidation energy was utilized to form delta mu H+. Comparison of the energetics revealed that the magnitudes of delta Hox (the enthalpy of the oxidation reaction) and delta Hm (the enthalpy of the formation of delta mu H+) in the two energy sources were comparable (-46 kcal/mol of ascorbate to -40 kcal/mol of D-lactate for delta Hox and 9.6 kcal/mol of ascorbate to 14 kcal/mol of D-lactate for delta Hm). Comparable and low value (about 1%) was also found in the free energy transfer (defined by delta Gm/delta Gox) from the oxidation reaction to the formation of delta mu H+. These results, in combination with the close values of delta mu H+ observed in the two systems, suggested that the characteristic of the created delta mu H+ was independent of the energy source. Examination of delta Hm might provide the information on the ratio of the number of protons produced, as 1 mol of two different electron donors was oxidized. The oxidation reaction in the presence of membrane vesicles was discussed.     

Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H

A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).     

Gradation of the magnitude of the electrochemical proton gradient in Mycoplasma cells

The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non-metabolizable analogue methyl alpha-D-glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+-dependent ATPase (phi (cHxN)2C H+) and the respective amplitudes of the components of delta mu H+. When Mycoplasma cells are incubated with 10 mM methyl alpha-D-glucoside, the amplitude of phi (cHxN)2C H+, of the electrical potential delta psi and of the chemical gradient delta pH become continuous functions of external glucose concentration within the limits of the non-energized and fully energized states. Analysis of the relationships between graduated amplitudes of delta psi, delta pH and phi (cHxN) 2C H+ show that the primary form of energy stored by a delta mu H+ generator is the electrical potential.     

Regulation of the heart mitochondrial respiration rate. Comparison of oxidation of succinate and NAD-dependent substrates]

Regulation of respiration at all rates between State 4 and State 3 was studied in heart mitochondria oxidizing FAD- and NAD-dependent substrates (succinate, pyruvate + + malate and palmitoylcarnitine). The creatine phosphokinase ADP-regenerating system was used which allows to fix the concentrations of extramitochondrial adenine nucleotides in such a way that the rate of respiration is controlled by mitochondrial processes alone. It was shown that respiration is controlled by delta mu(H+)-utilizing system within the respiration rate interval from State 4 till 70-80% of the maximal rate in State 3 (corresponding to physiological rates) both for NAD- and FAD-dependent substrates. The main step in the control of respiration near State 4 is proton leakage through the inner mitochondrial membrane, whereas in all the other parts of the mentioned interval this role is assigned to the adenine nucleotide translocator (ANT). The control coefficient for ANT is higher, while that of proton leakage is lower at the same relative rates of respiration with NAD-dependent substrates compared with succinate. These differences were found to be related to much higher values of the membrane potential generated at the same relative rates of succinate oxidation in comparison with the case with pyruvate + + malate. The contribution of delta mu(H+)-utilizing system to respiration control sharply decreases, whereas that of the delta mu(H+)-generating system increases at maximal rates of respiration near State 3. This phenomenon in more characteristic of succinate. In this case the control coefficient of ANT drops to zero, while that of succinate dehydrogenase rises to 0.7.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

DCCD-sensitive Na+-transport in the membrane vesicles of Halobacterium halobium

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.     

Factors determining the plasma-membrane potential of lymphocytes.

1. Lymphocytes from pig mesenteric lymph node have low permeability to K+ (Rb+), Na+ and Cl-. None of these ions is in Nernst equilibrium with the plasma-membrane potential (delta psi p). 2. delta psi p can be calculated from the transmembrane distribution of the permeant cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to abolish uptake into intracellular mitochondria. In normal culture medium delta psi p is 56 mV. 3. A similar potential is found in T-enriched pig cells and in mouse thymocytes. 4. The contribution of electrogenic (Na+ + K+)ATPase to delta psi p is about 7 mV. 5. The remainder of the lymphocyte delta psi p is a polyionic potential set up by K+ and Cl- with a permeability coefficient for Cl- of similar magnitude to that for K+.     

Some novel aspects of the relationship between the amino acid gradient and the sodium electrochemical gradient in mouse ascites tumour cells

Accumulation of 2-aminoisobutyrate by mouse ascites tumour cells was studied in circumstances where nigericin reversed the normal direction of the Na+ concentration gradient. The membrane potential (delta psi) was assayed using oxonol V as a voltage-sensitive probe. The amino acid gradient (delta mu A) that formed did not significantly exceed the likely magnitude of the Na+ electrochemical gradient when this was in the range 2-6 kJ mol-1. When delta-Na mu increased up to 11 kJ mol-1, delta mu A was almost constant at 7-8 kJ mol-1. The observations indicate that when delta psi is large changes in cellular [Na+] in the range 16-80 mM scarcely affect delta mu A.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force

Evidence is presented for a proportional relationship between the extramitochondrial phosphate potential (delta Gexp) and the proton-motive force (delta mu H+) across the mitochondrial membrane in rat-liver mitochondria oxidising succinate in State 4, when delta mu H+ is varied by addition of uncouplers or malonate. This relationship was found when precautions were taken to minimise interference with the determination of delta Gexp and delta mu H+ by intramitochondrial nucleotides, adenylate kinase activity, the quenching method, and delta mu H+-dependent changes in matrix volume. A non-proportional delta Gexp/delta mu H+ relationship was obtained when these precautions were omitted. Our results do not support mosaic protonic coupling, but are not necessarily in conflict with other localised coupling schemes.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

Studies on the mechanism of oxidative phosphorylation. Flow-force relationships in mitochondrial energy-linked reactions

The relationship between the steady-state level of membrane potential (delta psi) and the rates of energy production and consumption has been studied in mitochondria and submitochondrial particles. The energy-linked reactions investigated were oxidative phosphorylation (with NADH, succinate, and beta-hydroxybutyrate as respiratory substrates) and nucleoside triphosphate-driven transhydrogenation from NADH to NADP and uphill electron transfer from succinate to NAD. Results have shown the following. 1) Attenuation of the rates of the energy-producing reactions results in a parallel change in the rates of the energy-consuming reactions with little or no change in the magnitude of steady-state delta psi. 2) At low rates of energy production and consumption, steady-state delta psi decreases. However, this is due largely to the energy leak of the system which lowers static-head delta psi when the rate of energy production is slow. 3) When the rate of energy production and static-head delta psi are held constant, and the rate of energy consumption is diminished by partial inhibition or the use of suboptimal conditions (e.g. subsaturating substrate concentrations), then even a small decrease in the rate of energy consumption results in an upward adjustment of the level of steady-state delta psi. The lower the rate of energy input, the greater the upward adjustment of steady-state delta psi upon suppression of the rate of energy consumption. 4) The above results have been discussed with regard to the role of bulk-phase delta mu H+ or delta psi in the mitochondrial energy transfer reactions.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.     

Enthalpy changes in the formation of the proton electrochemical potential and its components

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.     

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.