Na+-driven ATP synthesis of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

Abstract: In vivo ATP synthesis of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1, derived from endogenous respiration, was examined. ATP was synthesized at both pH 6.5 and 8.5 after the start of the endogenous respiration by supplying O₂ to the anaerobic cell suspension. The ATP synthesis at pH 6.5, but not at pH 8.5, was completely inhibited by a H⁺ conductor, carbonylcyanide m -chlorophenylhydrazone (CCCP). The CCCP-resistant ATP synthesis at pH 8.5 was strongly inhibited by an inhibitor of the respiration-dependent primary Na⁺ pump, 2- n -heptyl-4-hydroxyquinoline N -oxide, and essentially required Na⁺. These results show that this bacterium synthesizes ATP at pH 6.5 by electrochemical potentials across the membrane Δ ∼ μ _H+, whereas at pH 8.5 by Δ ∼ μ _Na+ but not Δ ∼ μ _H+.     

Rb+ uptake by isolated pea mesophyll protoplasts in light and darkness

Rb⁺ uptake into protoplasts isolated from the mesophyll of Pisum sativum L. cv. Dan has been followed at intervals of a few minutes in the light and in the dark. The progress curve for uptake in the dark decreased in slope after about 7 min; in the light, by contrast, the slope increased. This effect was more pronounced at pH 7 than at pH 5.5. The pH profile for uptake in the dark rose with increasing pH: in the light the profile flattened, or even fell somewhat, between pH 5.5 and pH 6.5, then rose again. In the dark the proton uncoupler carbonyl cyanide m-chlorphenylhydrazone (CCCP) had little or no effect, either at pH 5.5 or at pH 7.4; in the light CCCP was strongly inhibitory, particularly at pH 7.4. Increasing concentrations of CCCP produced progressively more and more severe inhibition in the light, but in the dark produced a slight rise in uptake. The ATPase inhibitors quercetin, rutin and diethyl-stilbestrol, as well as arsenate, all depressed uptake in the light, particularly at higher pH Dark uptake was sensitive only at pH 5.5, not at pH 7.4. In marked contrast to the case of methyl-3 glucose, where protoplasts which were switched from light to dark took up sugar at the accelerated light rate for the first 7 min in the dark, a switch to darkness produced a Rb⁺ uptake rate below that for protoplasts held continuously in the dark. It is inferred that the mechanism of Rb⁺ uptake does not involve proton cotransport. Information regarding the membrane potential was obtained by following the distribution of tetraphenyl phosphonium (TPP⁺) between protoplasts and medium. The potential was more negative in the light than in the dark. It was also more negative at pH 7 than at pH 5 both in the light and in the dark. Treatment with CCCP produced no appreciable depolarization within the first 20 min, indicating thet the CCCP inhibition of Rb⁺ uptake in the light cannot be ascribed to a reduction in potential. An ATP-fueled K⁺ porter, or K⁺-H⁺ antiporter, seems the most likely explanation. The maintenance of the rising pH profile in the dark, despite the presence of a CCCP concentration which drastically inhibits light uptake, suggests that the profile does not depend on the operation of the proton pump.     

Energetics of galactose,proline, and glutamine transport in a cytochrome-deficient mutant of salmonella typhimurium

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the β-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca²⁺, Mg²⁺-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.     

Effects of Internal pH on the Acetylcholine Transporter of Synaptic Vesicles

Abstract: Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [³H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [¹⁴C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [³H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [³H]ACh. It exhibited K _m and V _max values of ∼200–500 µ M and 7–30 nmol [³H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP-dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 ± 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH ∼2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear. In vivo, the steep dependence of transport on the transmembrane pH gradient might serve to minimize leakage of ACh from the cytoplasm due to ACh transporter in the plasma membrane.     

Effect of light intensity on proton extrusion by roots of intact maize plants

Shading of maize plants ( Zea mays L. cv. Blizzard) reduced net H⁺ extrusion by roots and increased K⁺ release, whereas there was no significant effect on anion efflux in deionized water. With lower light intensity the concentrations of carbohydrates in the roots decreased, but ATP levels and energy charge remained unchanged. Also, shading raised the tissue pH of roots and made the cytoplasmic pH of root cells drop. There was a significant influence of light intensity on H⁺ uptake by roots from an acidified test solution and CCCP (carbonylcyanide-3-chlorophenylhydrazone)-in-duced H⁺ uptake was modified by shading.
It is concluded that low light intensity does not limit active H⁺ release by plasmalemma ATPase activity in the root cells, but that a reduced carbohydrate supply brings about a change in biochemical reactions which alter the membrane permeability for protons. An increased passive reflux of H⁺ into the cells rather than a reduced H⁺ ATPase activity explains the decrease of net H⁺ release by roots of intact maize plants under low light intensity.     

Proton and sucrose transport in isolated tonoplast vesicles from sugarcane stalk tissue

Tonoplast vesicles prepared from immature sugarcane ( Saccharum spp., hybrid cv. H65–7052) tissue and purified on a discontinuous dextran gradient take up sucrose. Uptake was stimulated by MgATP. Evidence that the mechanism is linked to proton transport is derived from "pH jump'data and from inhibition of ATP-stimulated sucrose transport by the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP) and by the proton-channel blocker of proton-linked ATPases. N. N '-dicyclo-hexylcarbodiimide (DCCD). A saturable phase of sucrose uptake was found at low substrate concentrations, and a linear phase characterized uptake at higher concentrations. Uptake was specific for sucrose, as demonstrated by competition experiments with various sugars. Sucrose uptake by the vesicle fraction was inhibited by KNO₃, protonophores and protein modifying reagents, whereas sodium orthovanadate had no effect. Overall, the evidence suggests an ATP-hydrolysis-dependent tonoplasl antiport for sucrose transport, although a more direct influence of ATP on conformational changes in relevant tonoplast proteins cannot be ruled out.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.     

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.     

An evaluation of N-phenyl-1-naphthylamine as a probe of membrane energy state in Escherichia coli

Colicin E1 and the uncoupler of oxidative phosphorylation, trifluoromethoxy-carbonylcyanidephenylhydrazone (FCCP), cause an increase in the fluorescence intensity of N-phenyl-1-naphthylamine bound to whole cells of Escherichia coli. It has been shown elsewhere that this fluorescence increase correlates well with de-energization. Addition of glucose causes a large cyanide-sensitive decrease of intensity, tentatively associated with energization, with the emission spectrum almost returning to the original trace with a peak at 417 nm. These data suggest that there may be a measurable competition between de-energization and energization of the cell membrane, and that the probe fluorescence intensity may be a general indicator of membrane energy level.

The conclusions reached about cellular energy level from measurements of the probe fluorescence intensity correlate partly (a, b below, not c) with the energy level assayed physiologically through rates of active transport: (a) FCCP is found to be a poor inhibitor of proline transport if cells are first incubated with glucose, showing either competition between the processes of energization and de-energization or an increase in the envelope permeability barrier to FCCP caused by glucose addition. (b) Cyanide blocks the fluorescence decrease caused by glucose and inhibits proline and serine transport, consistent with the decrease in probe fluorescence intensity indicating an increase in membrane energization. However, (c) it appears that the amplitude of the fluorescence intensity decrease caused by glucose addition in the presence of FCCP and colicin E1 greatly exaggerates the extent of real membrane energization. Glucose added after uncoupler can cause only a small increase, and after colicin, a negligible increase in the proline transport rate, indicating that the magnitude of the fluorescence intensity decrease after glucose addition is not a true measure of membrane energization, but rather seems to amplify this energization greatly. Glucose addition does not cause a decrease in fluorescence intensity in cells treated with EDTA to remove lipopolysaccharide and an apparent barrier to the probe.

The rotational relaxation time of the probe in intact cells appears to correlate somewhat better with the cellular energy level than does intensity.     

An electrogenic uniport mediates light-dependent Ca influx into intact spinach chloroplasts

Light-dependent Ca²⁺ influx into intact spinach chloroplasts, measured with the metallochromic indicator arsenazo III, is stimulated by uncouplers (FCCP, CCCP, nigericin) and inhibited by ruthenium red. The data presented demonstrate that light-dependent Ca²⁺ influx into chloroplasts is electrogenic and mediated by a uniport-type carrier. The characteristics of the carrier system are similar to those of the Ca²⁺ uniport of mitochondria.     

Calcium transport and ATPase activity in a microsomal vesicle fraction from corn roots

Abstract. An investigation has been made of methods for isolating membrane vesicles from corn ( Zea mays L.) roots active in calcium transport and K⁺-stimulated ATPase. Pretreating and grinding the roots at room temperature with EGTA and fusicoccin increases basal ATPase activity. Improvement in Ca²⁺ uptake requires isolation of a scaled vesicle fraction by the method of Sze(1980). Sorbitol is superior to sucrose as an osmoticant. The pH optimum for Ca²⁺ uptake is 7.5. whereas that for associated ATPase activity is 6.5. Calmodulin strongly stimulates Ca²⁺ uptake in a process little affected by uncouplers and ATPase inhibitors, but blocked by chlorpromazine. Fusicoccin gives less stimulation of Ca²⁺ uptake which is sensitive to uncouplers, and is dependent upon isolation with fusicoccin present. It appears that the sealed vesicle fraction may possess two Ca²⁺ transport systems: a calmodulin-activated Ca²⁺-transporting ATPase, and a Ca²⁺/H⁺ antiport coupled through the protonmotive force to a fusicoccin-stimulated H⁺-ATPase.     

Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Na+ pump functions

The growth of Vibrio alginolyticus and V. costicola, which possess respiration-dependent Na+ pumps, was highly resistant to the proton conductor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), in alkaline growth media, even though the membrane was rendered permeable to H+. The pH dependence of CCCP-resistant growth was similar to that of the Na+ pump. In contrast, Escherichia coli ML308-225 showed neither Na+ pump activity nor CCCP-resistant growth, even when grown in alkaline, Na+-rich media. These results suggest that certain bacteria possess the Na+ pump and are thus able to grow under the conditions where H+ circulation across the membrane does not take place. Moreover, V. alginolyticus growing in the presence of CCCP maintains normal levels of internal K+, Na+, and H+. The Na+ pump, therefore, makes the growth of these organisms resistant to CCCP by maintaining the intracellular cation environments.     

Toxicity of organotins towards cyanobacterial photosynthesis and nitrogen fixation

Abstract Inhibition of photosynthesis by a range of organotin compounds in Plectonema boryanum was concentration-dependent and decreased in the order tributyltin (Bu₃SnCl) > tripropyltin (Pr₃SnCl) ≥ dibutyltin (Bu₂SnCl₂) ≥ triphenyltin (Ph₃SnCl) > triethyltin (Et₃SnCl) > trimethyltin (Me₃SnCl) > monobutyltin (BuSnCl₃). IC₅₀ values were determined for the most toxic organotin species and varied from approximately 1.2 μM for Bu₃SnCl to approximately 13 μM for Ph₃SnCl. A similar order of inhibition of photosynthesis was observed in Anabaena cylindrica , although here IC₅₀ values were slightly lower (e.g. approximately 1 μM for Bu₃SnCl and 5 μM for Ph₃SnCl).Nitrogenase activity was generally more sensitive to inhibition by organotin compounds than photosynthesis in A. cylindrica and this was particularlyy evident for Bu₂SnCl₂; approximate IC₅₀ values for Bu₂SnCl₂ were 3 and 9 μM, as estimated by nitrogenase activity and photosynthesis, respectively. These results indicate that organotin compounds have the potential to inhibit cyanobacterial metabolism in aquatic systems.     

Uncouplers of oxidative phosphorylation can enhance a Fas death signal

Recent work suggests a participation of mitochondria in apoptotic cell death. This role includes the release of apoptogenic molecules into the cytosol preceding or after a loss of mitochondrial membrane potential DeltaPsim. The two uncouplers of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2, 4-dinitrophenol (DNP) reduce DeltaPsim by direct attack of the proton gradient across the inner mitochondrial membrane. Here we show that both compounds enhance the apoptosis-inducing capacity of Fas/APO-1/CD95 signaling in Jurkat and CEM cells without causing apoptotic changes on their own account. This amplification occurred upstream or at the level of caspases and was not inhibited by Bcl-2. The effect could be blocked by the cowpox protein CrmA and is thus likely to require caspase 8 activity. Apoptosis induction by staurosporine in Jurkat cells as well as by Fas in SKW6 cells was unaffected by CCCP and DNP. The role of cytochrome c during Fas-DNP signaling was investigated. No early cytochrome c release from mitochondria was detected by Western blotting. Functional assays with cytoplasmic preparations from Fas-DNP-treated cells also indicated that there was no major contribution by cytochrome c or caspase 9 to the activation of effector caspases. Furthermore, an increase of rhodamine-123 uptake into intact cells, which has been explained by mitochondrial swelling, occurred considerably later than the caspase activation and was blocked by Z-VAD-fmk. These data show that uncouplers of oxidative phosphorylation can presensitize some but not all cells for a Fas death signal and provide information about the existence of separate pathways in the induction of apoptosis.     

Rate limiting factors in leaf photosynthesis. II. Electron transport

Increased scattering of a weak 535 nm measuring beam which indicates the light-dependent formation of a transthylakoid proton gradient in leaves was used to examine the role of the electron-transport chain in limiting photosynthetic carbon assimilation. The proton gradient is supported by electron flux and indicates thylakoid energization. In CO₂-free air, half saturation of thylakoid energization was observed at intensities of red light ranging from 2 to 50 W·m⁻² in different plant species. The differences were attributed to different carbohydrate availability for energy-consuming photorespiratory processes when external CO₂ was absent. Thylakoid energization of shade leaves (Asarum, Fagus) was saturated at lower light intensities than that of sun leaves (Phaseolus, Fagus). When photorespiratory carbohydrate oxidation was suppressed by decreasing the O₂ concentration from 21 to 2% in the absence of CO₂, thylakoid energization saturated at lower light intensities than in CO₂-free air. CO₂ decreased thylakoid energization particularly at low light intensities. Under high intensity illumination, however, thylakoid energization was remarkably high even in the presence of saturating CO₂. Apparently, electron transport was capable of maintaining the energy status of the photosynthetic apparatus at a high level even when photosynthetic carbon fluxes were maximal. This suggests that electron transport is less important in limiting photosynthesis than previously thought.     

Key-word system of indexing

The effects of uncouplers of photophosphorylation on the P-S₁ transient of the fluorescence induction in darkadapted intact chloroplasts of Bryopsis maxima were studied to examine the mechanism of light-dependent regulatory changes in electron transport. (1) Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nigericin slowed down the fluorescence quenching from P to S₁, whereas the transient was significantly accelerated by the addition of NH₄Cl and methylamine. (2) The P-S₁ decline was slowed down at low pH of the suspending medium, suggesting sensitivity of the transient to the stroma pH. The inhibitory effect of nigericin was markedly enhanced at low pH and at low KCl concentrations, whereas the ionophore stimulated the transient at high pH and at high KCl concentrations. Similar results were obtained on the combined addition of CCCP and valinomycin. (3) Nigericin and the CCCP-valinomycin couple altered the internal pH of intact chloroplast through the H⁺-K⁺ exchange across the outer limiting membrane. The fluorescence decline was rapid at alkaline internal pH but was suppressed with lowering internal pH below 8.0 (4) A similar internal pH dependence of the transient was obtained when the internal pH was changed by the addition of NH₄Cl and acetate. (5) It is proposed that the photoactivation of electron transport is regulated by the stroma pH. The progress of the photoactivation is slow at acidic or neutral pH but is significantly accelerated by light-induced alkalinization near the light-regulation site of electron transport located on the outer surface of the thylakoid membrane.     

The influence of uncouplers on facilitated diffusion of sorbose in Saccharomyces cerevisiae

Sorbose uptake in Saccharomyces cerevisiae, strain Delft 1, proceeds via mediated passive transport. In the cell sorbose is distributed in at least two compartments. Efflux studies showed that sorbose uptake in one of these compartments is not readily reversible. Uncouplers of oxidative phosphorylation inhibit both transport velocity and steady-state uptake level. It could be shown that these two effects are caused by different modes of action of the uncouplers. None of these two effects could be ascribed to changes of the electrochemical H⁺ gradient or of the intracellular pH. It is suggested that the inhibition of uptake velocity is caused by binding of the uncoupler to the sorbose translocator, thus lowering the transport activity. The uncoupler binding site is probably located at the intracellular fragment of the carrier. The second effect, reduction of the steady-state uptake level, is probably due to blocking of sorbose influx into the compartment that exhibits poor reversibility.     

Artificially induced active transport of amino acid driven by the efflux of a sugar via a heterologous transport system in de-energized Escherichia coli.

Consistent with the model of an H+ cotransport, amino acid uptake can be driven by a proton gradient generated by an efflux of sugar when the normal energy sources are suppressed. Heterologous countertransport is completely inhibited by uncouplers unlike homologous countertransport. Positive coupling was obtained with methyl thiogalactoside/proline, methyl thiogalactoside/phenylalanine, gluconate/proline; however, the poor coupling efficiency suggests a more complex sequence of reactions.     

Energy transduction in vesicles of the methanogenic strain Gö1

Abstract Everted vesicles of the methanogenic strain Gö1 synthesized ATP in response to methanogenesis from methyl-coenzyme M and H₂. Simultaneously, a transmembrane pH gradient (ΔpH) was generated as evident from fluorescence quenching of acridine orange. Protonophorous uncouplers prevented ΔpH generation and ATP synthesis, but did not affect methanogenesis. The ATP synthase inhibitor diethylstilbestrol (DES) inhibited ATP synthesis but had no effect on methanogenesis and on ΔpH formation, indicating the essential role of the transmembrane proton potential in ATP synthesis. Progress has also been made in assigning specific functions to membrane components in methanogenesis from methyl-CoM and H₂. Separation of cell extracts into cytoplasmic and membrane fraction revealed an essential role of membrane-bound components in electron transfer: methanogenesis catalyzed by the cytoplasmic fraction from strain Gö1 was stimulated several fold by membranes from various methanogens. This stimulation was prevented if the membranes had been treated with oxidants (O₂, K₃[Fe(CN)₆]) or SH reagents (Ag⁺, p -chloromercuribenzoate, iodoacetamide) pointing to the involvement of functional SH groups in methanogenesis from methyl-CoM and H₂.     

Effects of amiodarone on K+, internal pH and Ca2+ homeostasis in Saccharomyces cerevisiae

In this study, amiodarone, at very low concentrations, produced a clear efflux of K⁺. Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K⁺ efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca²⁺ and H⁺, the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca²⁺ concentration, as well as the decreased internal pH. The Δ tok1 and Δ nha1 mutations resulted in a smaller effect of amiodarone, and Δ trk1 and Δ trk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K⁺, through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.