Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics)

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.     

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little ΔpH during growth) with an electrical potential of ?127 mV in growth medium and ?105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

Import of glyoxysomal malate dehydrogenase precursor into glyoxysomes: A heterologous in-vitro system

A heterologous in-vitro system is described for the import of the precursor to glyoxysomal malate dehydrogenase from watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) cotyledons into glyoxysomes from castor-bean (Ricinus communis L.) endosperm. The 41-kDa precursor is posttranslationally sequestered and correctly processed to the mature 33-kDa subunit by a crude glyoxysomal fraction or by glyoxysomes purified on a sucrose gradient. The import and the cleavage of the extrasequence is not inhibited by metal chelators such as 1,10-phenanthroline and ethylenediaminetetraacetic acid. Uncouplers (carbonylcyanide m-chlorophenylhydrazone), ionophores (valinomycin), or inhibitors of oxidative phosphorylation (oligomycin) and ATP-ADP translocation (carboxyatractyloside) do not interfere, thus indicating the independence of the process of import by the organelle from the energization of the glyoxysomal membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacctic acid - gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride     

Evidence for Endogenous Cyclic Photophosphorylation in Intact Chloroplasts: CO(2) Fixation with Dihydroxyacetone Phosphate

This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix ¹⁴CO₂ when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial ¹⁴CO₂ fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of ¹⁴CO₂ fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no ¹⁴CO₂ fixation occurred when 3-phosphoglycerate was used. ¹⁴CO₂ fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5′-phosphate. Low concentrations of O₂ (25-50 micromolar) stimulated ¹⁴CO₂ fixation, but the activity decreased with increasing O₂ concentrations. The fixation of ¹⁴CO₂ in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.     

Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

Surface potential on the periplasmic side of the photosynthetic membrane of Rhodopseudomonas sphaeroides

Membrane surface potential on the periplasmic side of the photosynthetic membrane was estimated in cells, spheroplasts and chromatophores of Rhodopseudomonas sphaeroides. When the membrane potential (potential difference between bulk aqueous phases) was kept constant in the presence of carbonylcyanide m-chlorophenylhydrazone, addition of salt to a suspension of cells or spheroplasts induced a red shift in the carotenoid absorption spectrum which indicated a change in the intramembrane electrical field. The spectral shift is explained by a rise in electrical potential at the outside surface of the photosynthetic membrane due to a decrease in extent of the negative surface potential.The spectral shift occurred in the direction opposite to that in chromatophores, indicating that the sidedness of the membrane of cells or spheroplasts is opposite to that of chromatophores. The dependences of the extent of the potential change on concentration and valence of cations of salts agreed with the Gouy-Chapman relationship on the electrical diffuse double layer. The charge density on the periplasmic surface of the photosynthetic membrane was estimated to be ?2.9 · 10^?3 elementary charge per Ų, while that on the cytoplasmic side surface was calculated as ?1.9 · 10^?3 elementary charge per Ų (Matsuura, K., Masamoto, K., Itoh, S. and Nishimura, M. (1979) Biochim. Biophys. Acta 547, 91–102). Surface potential on the periplasmic side of the photosynthetic membrane was estimated to be about ?50 mV at pH 7.8 in the presence of 0.1 M monovalent salt.     

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K_m for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H₂ adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).     

Suppression of Carotenoid Photobleaching by Kaempferol in Isolated Chloroplasts

The effects of kaempferol on carotenoid photobleaching wereexamined using chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone(CCCP). Kaempferol suppressed carotenoid photobleaching withoutaffecting electron transfer reactions. Half-maximal suppressionwas observed at about 10 µM. Kaempferol was photooxidizedby CCCP-poisoned chloroplasts, as observed by its bleachingat 380 nm. Ascorbate inhibited the oxidation of kaempferol.Under anaerobic conditions, kaempferol did not affect the photobleachingof carotenoid. Other fiavonols, quercetin and its glycosides,also suppressed the carotenoid photobleaching. The results suggestthat flavonols act as antioxidants in illuminated chloroplastsunder aerobic conditions. (Received February 22, 1982; Accepted May 14, 1982)     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca²⁺-activated ATPase activity could remain unaltered.Gramicidin stimulated calcium uptake irrespective of the presence of a K⁺ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K⁺ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates.Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore.After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca²⁺-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca²⁺-activated ATPase protein.     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.     

The surface and membrane potentials of chromatophores of photosynthetic bacteria as studied by carotenoid absorbance changes

The change in surface potential induced by addition of mono- or divalent cations to a chromatophore suspension was monitored by carotenoid absorbance changes (a probe which is intrinsic to the membrane). The change in carotenoid absorbance elicited by an alteration of the surface potential is strongly dependent on the presence of ionophores; the absorbance changes (due to addition of MgCl₂) in the presence of valinomycin or gramicidin are larger than those in the presence of carbonyl cyanide m-chlorophenylhydrazone or cabonyl cyanide p-trifluoromethoxyphenylhydrazone. These differences in carotenoid absorbance change reflect the degree in which the membrane resistance has been shunted. Gramicidin or high concentrations of valinomycin (10⁻⁶ M) appear to be sufficiently effective as shunt in order that the totality of the change in external surface potential is seen as an intramembrane potential difference as sensed by the carotenoids. It is also shown that the decay of the carotenoid changes induced by the addition of salt to the medium is a measure of the intrinsic permeability of the chromatophore membrane for the added cation.     

Transport of 1-aminocyclopropane-1-carboxylic acid into isolated maize mesophyll vacuoles

Intracellular transport of the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC) can change the ACC concentration in cell compartments and impact ethylene biosynthesis. Transport of ACC into isolated maize ( Zea mays L.) mesophyll vacuoles was studied by silicon layer flotation filtering. The transport of ACC across the tonoplast was stimulated 2. 4- to 8. 1-fold by 5 m M MgATP, showed saturation kinetics with an apparent K_m for ACC of 20 μ M , and was optimal at 25°C. Transport of ACC was sensitive to the pH of the medium, falling as external pH rose. Effectors known to inhibit proton-translocating ATPases (N, N-dicyclohexylcarbodiimide) and to collapse the electrical (thiocyanate, valinomycin) and chemical (carbonylcyanide m -chlorophenylhydrazone, gramicidin) potential gradients for protons across the tonoplast all reduced ACC transport. The nonhydrolyzable MgATP analog. Mg adenylyl-imidodiphosphate, stimulated ACC transport as effectively as MgATP. Other nucleotides (MgADP, MgCTP, MgUTP, MgGTP) and MgPP_i had little or no effect. These results suggest that ACC uptake into isolated maize mesophyll vacuoles is carrier mediated, is dependent upon an electrochemical potential gradient for protons and is specifically regulated, but not necessarily energized, by MgATP     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H+, K+ and Na+ in phospholipid vesicles

Rates of M⁺/H⁺ exchange (M⁺=K⁺, Na⁺) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M⁺ and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M⁺ dissociation constant, K_M, of the M⁺ bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H⁺ bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M⁺ and an anion) by forming 1:2 complexes with valinomycin–M⁺ or nonactin–M⁺. (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K_M increases. The decreases/increases in K_M mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

The electric unit size of thylakoid membranes.

The size of the function unit of electrical events in thylakoid membranes was estimated by the minimum amount of gramicidin needed to discharge the flash light generated electrical potential difference. Early flash spectroscopic measurements have indicated that a single gramicidin dimer operates on an electrical function unit containing at least 2 x 10(5) chlorophyll molecules. In this study we present gramicidin titrations with more intact thylakoid preparations which revealed a more than hundred-fold greater lower limit for the electric unit size, namely 5 x 10(7) chlorophyll molecules. It is conceivable that the whole complicated thylakoid structure inside a chloroplast constitutes a single electric unit. It comprises more than 2 x 10(8) chlorophyll molecules in an area of more than 400 microns 2.     

The effect of amine structure on complexation with lasalocid in model membrane systems: I. Identification of charged complexes in lipid bilayer membranes

The electrical properties of X-537A (lasalocid) doped lipid bilayer membranes were studied in the presence of a series of nine biogenic amines which contain β-phenylethylamine as the basic structural unit. The ionophore antibiotic was found to form charged complexes within the membrane during the transport of some of the amines. The dependence of membrane conductance on the concentration of ionophore and amine was studied. The amines are divided into three classes according to the nature of the complexes formed: (1) charged complex involving two ionophores (phenylephrine, metanephrine, and amphetamine); (2) charged complex containing three ionophores (dopamine, norepinephrine and epinephrine); and (3) no charged species formed (p- and m-tyramine and β-phenylethylamine).     

The Effect of Metabolic Inhibitors, Sugars and Fusicoccin on the Electrical Potential Difference Arising Across an Intact Chenopodium Rubrum L. Plant

An analysis of the effect of metabolic inhibitors, sugars, and fusicoccin on the trans-plant electrical potential difference arising across one-week-old green or herbicide-treated Chenopodium rubrum L. plants was performed. The substances were applied either to the solution bathing the root or in the form of drops to the stem. The respiratory inhibitors (KCN and salicylhydroxamic acid), sulfhydryl agents (N-ethylmaleimide and p-chloromercuribenzene sulfonic acid) and proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) affected the electrical potential, the kinetics of the induced changes varying with different inhibitors and site of application. None of the applied sugars (sucrose, glucose or sorbitol), ATPase stimulator fusicoccin or inhibitor vanadate exerted any appreciable effect on the electrical potential. An effect of sucrose could be observed in the case of its application immediately following de-rooting, especially in the case of herbicide-treated plants. These results we explain by non-participation of the sucrose transporter or the proton ATPase in the generation of the electrical potential difference across intact plants (apoplast-apoplast configuration).