Oxonol VI Research Articles

Functional Identification of H+-ATPase and Na+/H+ Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte Suaeda altissima

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H+-ATPase in the PM of the root cells of S. altissima. The addition of Na+ and Li+ ions to the outer medium resulted in dissipation of ΔpH preformed by the H+-ATPase, which indicates the presence in PM of the functionally active Na+/H+ antiporter. The results are discussed with regard to involvement of the Na+/H+ antiporter and the PM H+-ATPase in loading Na+ ions into the xylem of S. altissima roots.

Interaction between DMPC liposomes and HM-PNIPAM polymer

The interactions of hydrophobically-modified poly-( N-isopropylacrylamides) (HM-PNIPAM) and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated by the effect of the polymer on the binding of a fluorescent dye, oxonol VI, to DMPC vesicles, and on its diffusion across the membrane. On mixing with the vesicles, the dye exhibits an increase in fluorescence, which occurs in a two-stage process. The process was monitored by stopped-flow fluorescence spectrophotometry. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid stage seems to be due to the second-order binding of the dye to the lipid membrane, a process that is almost diffusion-controlled, whereas the slow process is attributed to movement of the dye within the membrane phase. The polymer did not significantly affect the rate constant of the binding step, but it slowed down slightly the dissociation process of the dye from the membrane. However, the polymer affected the second stage, causing an increase in the reciprocal of its relaxation time, which suggests that the polymer makes the vesicle membrane more fluid.

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H +-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δ ψ) generated by the Schizosaccharomyces pombe plasma membrane H +-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δ ψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δ ψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H +-ATPase generates Δ ψ of about 160 mV on the vesicle membrane. The generated Δ ψ was stable at least over tens of minutes. An influence of the H + membrane permeability on the Δ ψ buildup was demonstrated by manipulating the H + permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H +-ATPase-mediated Δ ψ buildup.

Physiological parameters of gravitaxis in the flagellate Euglena gracilis obtained during a parabolic flight campaign

The unicellular freshwater flagellate Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitaxis. Previous experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism in which changes of the internal calcium concentration and the membrane potential play an important role. In a recent parabolic flight experiment on board an aircraft (ESA 29th parabolic flight campaign), changes of graviorientation, membrane potential and the cytosolic calcium concentration upon changes of the acceleration (between 1 x g(n), 1.8 x g(n), microgravity) were monitored by image analysis and photometric methods using Oxonol VI (membrane potential) and Calcium Crimson (cytosolic calcium concentration). The parabolic flight maneuvers performed by the aircraft resulted in transient phases of 1.8 x g(n) (about 20 s), microgravity (about 22 s) followed by 1.8 x g(n) (about 20 s). A transient increase in the intracellular calcium concentration was detected from lower to higher accelerations (1 x g(n) to 1.8 x g(n) or microgravity to 1.8 x g(n)). Oxonol VI-labeled cells showed a signal, which indicates a depolarization during the transition from 1 x g(n) to 1.8 x g(n), a weak repolarization in microgravity followed by a rapid repolarization in the subsequent 1 x g(n) phase. The results show good coincidence with observations of recent terrestrial and space experiments.

Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis.

Characterization of Isolated Acidocalcisomes of Trypanosoma cruzi

The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000 x g) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca(2+) uptake (Ca(2+)-ATPase) activity was detectable in the isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified, and the rate of Ca(2+) uptake increased. Use of the indicator Oxonol VI showed that V-H(+)-PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H(+)-PPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H(+)-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H(+)-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H(+)-ATPase and V-H(+)-PPase activities are present in the same Ca(2+)-containing compartment.

Evidence for the role of a Na(+)/HCO(3)(-) cotransporter in trout hepatocyte pHi regulation.

The mechanisms of intracellular pH (pHi) regulation were examined in hepatocytes of the rainbow trout Oncorhynchus mykiss. pHi was monitored using the pH-sensitive fluorescent dye BCECF, and the effects of various media and pharmacological agents were examined for their influence on baseline pHi and recovery rates from acid and base loading. Rates of Na(+) uptake were measured using (22)Na, and changes in membrane potential were examined using the potentiometric fluorescent dye Oxonol VI. The rate of proton extrusion following acid loading was diminished by the blockade of either Na(+)/H(+) exchange (using amiloride) or anion transport (using DIDS). The removal of external HCO(3)(-) and the abolition of outward K(+) diffusion by the channel blocker Ba(2+) also decreased the rate of proton extrusion following acid load. Depolarization of the cell membrane with 50 mmol l(-)(1) K(+), however, did not affect pHi. The rate of recovery from base loading was significantly diminished by the blockade of anion transport, removal of external HCO(3)(-) and, to a lesser extent, by blocking Na(+)/H(+) exchange. The blockade of K(+) conductance had no effect. The decrease in Na(+) uptake rate observed in the presence of the anion transport blocker DIDS and the DIDS-sensitive hyperpolarization of membrane potential during recovery from acid loading suggest that a Na(+)-dependent electrogenic transport system is involved in the restoration of pHi after intracellular acidification. The effects on baseline pHi indicate that the different membrane exchangers are tonically active in the maintenance of steady-state pHi. This study confirms the roles of a Na(+)/H(+) exchanger and a Cl(-)/HCO(3)(-) exchanger in the regulation of trout hepatocyte pHi and provides new evidence that a Na(+)/HCO(3)(-) cotransporter contributes to pHi regulation.

Electrogenicity of the Na +-ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H + countertransport

Sodium accumulation by the Na +-ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by Δ Ψ generation across the vesicle membrane (positive inside) and H + efflux from the vesicle lumen. Na + accumulation was assayed with 22Na +; Δ Ψ generation was detected by recording absorption changes of oxonol VI; H + efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP-dependent Na + uptake and H + ejection were increased by the H + ionophore carbonyl cyanide m-chlorophenylhydrazone (ClCCP) while Δ Ψ was collapsed. The lipophilic anion tetraphenylboron ion (TPB −) inhibited H + ejection from the vesicles and abolished Δ Ψ. Based on the effects of ClCCP and TPB − on H + ejection and Δ Ψ generation, the conclusion was drawn that H + countertransport observed during Na +-ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na + translocation across the vesicle membrane. Increasing Na + concentrations stimulated H + efflux and caused the decrease in the Δ Ψ observed, thus indicating that Na + is likely a factor controlling H + permeability of the vesicle membrane.

Electrophysiological Study with Oxonol VI of Passive NO 3− Transport by Isolated Plant Root Plasma Membrane

In contrast to animal cells, plant cells contain ∼5–50 mM nitrate in cytosol and vacuole. The lack of specific spectroscopic probes, or suitable isotopes, impedes in vitro studies of NO 3 − transport. Reconstitution of root cell plasma membrane (PM) proteins in mixed soybean lipid:egg phosphatidylcholine allowed for the generation of large K +-valinomycin diffusion potentials ( E m), monitored with the oxonol VI dye. Nevertheless, E m was restricted to ∼130 mV by capacitor properties of biological membranes. This caused an increasing discrepancy at higher K +-Nernst potentials used for calibration. Therefore, E m was determined directly from the fluorescence of the dye free in buffer, bound at zero E m, and bound upon E m generation. Then, an electrophysiological analysis of the NO 3 −-dependent dissipation rate of E m gave the net passive flux ( J N) and the permeability coefficient to NO 3 − ( P N). The plant root cell PM exhibited a strikingly large P N (higher than 10 −9 m s −1) at high E m (90–100 mV) and pH 6.5. At low E m (50–60 mV) and pH 7.4, P N decreased by 70-fold and became similar to that of the lipid bilayer. This agreed with the previous observation that 15 mM NO 3 − short-circuits the plant root PM H +-ATPase at its optimal pH of 6.5.

Ca2+ Transport by Reconstituted Synaptosomal ATPase Is Associated with H+ Countertransport and Net Charge Displacement

The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.

In vitro study of passive nitrate transport by native and reconstituted plasma membrane vesicles from corn root cells

Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H +)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K + diffusion gradient. Consequently, the H +-pumping activity of the (H +)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO 3 − to electrically short-circuit the (H +)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable ( K m≈5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO 3 −>I −≈ClO 3 −≈Br −>Cl −≈NO 2 −>Iminodiacetate≈SO 4 2−). Passive NO 3 − transport was also determined, independently of the (H +)ATPase, from the NO 3 −-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K m for NO 3 − and should be suitable to continue the functional and biochemical characterization of the NO 3 − transport system.

Measurement of the Membrane Potential Generated by Complex I in Submitochondrial Particles

To investigate the energy-conserving function of the NADH:ubiquinone reductase (complex I), we have selected oxonol VI [bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol] as the most sensitive probe for measuring the reactions of membrane potential generation in submitochondrial particles. Calibration of the oxonol signals with potassium diffusion potentials shows a non-linear response after a threshold around -50 mV. Thermodynamic evaluations indicate that the upper limit of the oxonol response to the potential generated by complex I is around -220 mV, which is close to the maximal protonmotive force in coupled submitochondrial particles. NADH addition to particles in which ubiquinol oxidation is blocked by inhibitors of other respiratory complexes generates oxonol signals corresponding to membrane potentials of -130 to -180 mV. These signals are produced by about four turnovers of the complex reducing endogenous ubiquinone (i.e. non-steady-state conditions) and are equivalent to a charge separation similar to that of the antimycin-sensitive reactions of ubiquinol:cytochrome c reductase (complex III). The transient oxonol signals under non-steady-state conditions are thus informative of crucial steps in the electrogenic reactions catalyzed by complex I. The possible nature of these electrogenic reactions is discussed in relation to proposed mechanisms for complex I.

Estimation of membrane potential deltapsi in reconstituted plasma membrane vesicles using a numerical model of oxonol VI distribution.

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference deltapsi across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. deltapsi was generated by the H+-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K+ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding deltapsi in ATP-energized PMV of 80 mV. The described model treatment to estimate deltapsi may be applicable for other reconstituted membrane systems.

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H+-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.

Evidence for the presence of two rotenone-insensitive NAD(P)H dehydrogenases on the inner surface of the inner membrane of potato tuber mitochondria

Submitochondrial particles were isolated from potato ( Solanum tuberosum L.) tubers. The latency of cytochrome- c oxidase and succinate dehydrogenase indicated that they were 90% inside-out. The inability of the submitochondrial particles to form a membrane potential inside negative as monitored by safranine absorbance changes and their ability to form a large membrane potential inside positive as monitored by oxonol VI absorbance changes confirmed this sidedness. Through the use of rotenone to inhibit Complex I, and diphenyleneiodonium to inhibit both Complex I (by binding to the FMN in the active site) as well as rotenone-insensitive NADPH oxidation, it was possible to distinguish three different NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane: (1) a rotenone-sensitive, diphenyleneiodonium-sensitive, Ca 2+-independent enzyme which prefers NADH as the substrate, i.e., Complex I; (2) a rotenone-insensitive, diphenyleneidoonium-sensitive, Ca 2+-dependent NAD(P)H dehydrogenase; (3) a rotenone-insensitive, diphenyleneiodonium-insensitive, Ca 2+-independent NADH dehydrogenase. All three enzymes were linked to the electron transport chain before Complex III as shown by antimycin A sensitivity and to proton pumping as shown by the generation of a membrane potential. The possible significance of these three enzymes for the function of the mitochondrion in the plant cell is discussed.

Unreliability of carotenoid electrochromism for the measure of electrical potential differences induced by ATP hydrolysis in bacterial chromatophores

ATP hydrolysis induces the activation of the proton ATPase in chromatophores of Rhodobacter capsulatus supplemented with nigericine and 50 mM K + (i.e. when ΔpH < 0.2 units). The value of transmembrane electric potential (Δϕ) driving this activation was measured using three different approaches: carotenoid electrochromism, uptake of SCN − and responses of the dye oxonol VI. The value of Δϕ calculated from the SCN − uptake, on the basis of an internal volume determined experimentally, was about 140 mV, while that indicated by the electrochromic signal ranged between 35 and 70 mV. Only the value indicated by SCN - distribution is consistent with the energetic requirement for the activation of H +-ATPase.

Use of two oxonols and a fluorescent tetrazolium dye to monitor starvation of Escherichia coli in seawater by flow cytometry

Flow cytometry was used to study starvation of Escherichia coli in artificial seawater using two membrane potential sensitive dyes and redox dye. The oxonols, DiBAC4(3) and oxonol VI, enabled assessment of energization states. Parallel use of these two dyes suggests that oxonol VI is the more useful for detection of membrane depolarization. The NADPH reducibility of 5-cyano-2,3-ditolyl tetrazolium chloride can be used to estimate loss of membrane integrity during survival in seawater.

ADP controls the electrogenicity of Na/Na exchange catalyzed by dog kidney Na,K-ATPase proteoliposomes

Sodium pump mediated Na/Na exchange was studied using Na +-loaded proteoliposomes prepared from dog kidney Na,K-ATPase. Measurements of both 22Na + influx and pump-generated electrical potentials were carried out, the latter using the anionic dye, oxonol VI. In the presence of ATP, the formation of a strophanthidin-sensitive membrane potential confirms that Na/Na exchange associated with ATP hydrolysis can be electrogenic depending on the source of the enzyme. With the addition of varying concentrations of ADP, electrogenic exchange is progressively inhibited and replaced by electroneutral exchange. ADP is equally effective in activating (ATP + ADP)-dependent electroneutral exchange. With sufficient ADP, electrogenic Na/Na exchange is completely replaced by electroneutral exchange.

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb 561. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb α-band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.

Liposomes in Reconstitution of Ion-Pumps. Electrogenic Properties of the Na+,K+-Atpase and the Sarcoplasmic Ca2+-Atpase

AbstractAny electrogenic ion-pump carrying a net-current during turnover is an electromotive device creating a transmembrane potential in tight vesicles, which can be detected by the potential sensitive fluorochrome oxonol VI. For the Na+,K+-ATPase the coupling ratio Na+:K+:ATP during physiological Na+:K+-exchange is 3:2:1, giving one positive net-charge translocated per ATP split. The same stoichiometry is found for the electrogenic Na+:Na+-exchange, whereas during uncoupled Na+-efflux this net-charge stoichiometry changes to three, in accordance with a transport stoichiometry 3:0:1. By inducing internal electrostatic potentials in the proteoliposome bilayer using the hydrophobic ions TPB or TPP+ it could be shown that the backreaction which normally translocates K+ changes from electroneutral to electrogenic during the uncoupled Na+-efflux where no ions are returned.For Ca2+-transport a stoichiometry of close to, but lower than 2 Ca2+-ions per ATP split is found. Recent findings indicate that protons ma...