On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells

The potential difference across the thylakoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illumination. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3–3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.     

Evidence for A Ca2+ gradient across the plasma membrane of wheat protoplasts

Fluxes of Ca²⁺ across the plasma membrane of isolated wheat protoplasts have been measured both as net accumulation and as uptake under steady-state conditions. The ATPase inhibitors, orthovanadate and diethylstibesterol, and the divalent cation ionophore, A23187, were all found to enhance net Ca²⁺ accumulation by protoplasts. The uptake of Ca²⁺ under steady-state conditions was also stimulated by A23187 but relatively unaffected by a range of plant hormones or by red or far red light. Light treatments were compared to dark controls with protoplasts isolated from etiolated wheat.The results suggest that plant cells maintain a Ca²⁺ gradient across their plasma membrane but it appears not to be under phytochrome control.     

Photochemical and Nonphotochemical Fluorescence Quenching Processes in the Diatom Phaeodactylum tricornutum

Nonphotochemical fluorescence quenching was found to exist in the dark-adapted state in the diatom Phaeodactylum tricornutum. Pretreatment of cells with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) or with nigericin resulted in increases in dark-adapted minimum and maximum fluorescence yields. This suggests that a pH gradient exists across the thylakoid membrane in the dark, which serves to quench fluorescence levels nonphotochemically. The physiological processes involved in establishing this proton gradient were sensitive to anaerobiosis and antimycin A. Based on these results, it is likely that this energization of the thylakoid membrane is due in part to chlororespiration, which involves oxygen-dependent electron flow through the plastoquinone pool. Chlororespiration has been shown previously to occur in diatoms. In addition, we observed that cells treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea exhibited very strong nonphotochemical quenching when illuminated with actinic light. The rate and extent of this quenching were light-intensity dependent. This quenching was reversed upon addition of CCCP or nigericin and was thus due primarily to the establishment of a pH gradient across the thylakoid membrane. Preincubation of cells with CCCP or nigericin or antimycin A completely abolished this quenching. Cyclic electron transport processes around photosystem I may be involved in establishing this proton gradient across the thylakoid membrane under conditions where linear electron transport is inhibited. At steady state under normal physiological conditions, the qualitative changes in photochemical and nonphotochemical fluorescence quenching at increasing photon flux densities were similar to those in higher plants. However, important quantitative differences existed at limiting and saturating intensities. Dissimilarities in the factors that regulate fluorescence quenching mechanisms in these organisms may account for these differences.     

Kinetics of electron transfer through the respiratory chain

We show that the rate at which electrons pass through the respiratory chain in mitochondria and respiring prokaryotic cells is described by the product of three terms, one describing electron donation, one acceptance, and a third, the thermodynamic drive. We apply the theory of nonequilibrium thermodynamics in the context of the chemiosmotic model of proton translocation and energy conservation. This approach leads to a closed-form expression that predicts steady-state electron flux as a function of chemical conditions and the proton motive force across the mitochondrial inner membrane or prokaryotic cytoplasmic membrane. The rate expression, derived considering reverse and forward electron flow, is the first to account for both thermodynamic and kinetic controls on the respiration rate. The expression can be simplified under specific conditions to give rate laws of various forms familiar in cellular physiology and microbial ecology. The expression explains the nonlinear dependence of flux on electrical potential gradient, its hyperbolic dependence on substrate concentration, and the inhibiting effects of reaction products. It provides a theoretical basis for investigating life under unusual conditions, such as microbial respiration in alkaline waters.     

Calcium efflux and cycling across the synaptosomal plasma membrane.

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.     

The Ventral Photoreceptor Cells of Limulus : III. A voltage-clamp study

In the dark, the ventral photoreceptor of Limulus exhibits time-variant currents under voltage-clamp conditions; that is, if the membrane potential of the cell is clamped to a depolarized value there is an initial large outward current which slowly declines to a steady level. The current-voltage relation of the cell in the dark is nonlinear. The only ion tested which has any effect on the current-voltage relation is potassium; high potassium shifts the reversal potential towards zero and introduces a negative slope-conductance region. When the cell is illuminated under voltage-clamp conditions, an additional current, the light-induced current, flows across the cell membrane. The time course of this current mimics the time course of the light response (receptor potential) in the unclamped cell; namely, an initial transient phase is followed by a steady-state phase. The amplitude of the peak transient current can be as large as 60 times the amplitude of the steady-state current, while in the unclamped cell the amplitude of the peak transient voltage never exceeds 4 times the amplitude of the steady-state voltage. The current-voltage relations of the additional light-induced current obtained for different instants of time are also nonlinear, but differ from the current-voltage relations of the dark current. The ions tested which have the greatest effect on the light-induced current are sodium and calcium; low sodium decreases the current, while low calcium increases the current. The data strongly support the hypothesis that two systems of electric current exist in the membrane. Thus the total ionic current which flows in the membrane is accounted for as the sum of a dark current and a light-induced current.     

Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.     

Glucose determination by means of a new reactor/sensor system operating under non-isothermal conditions

The dynamic and steady-state responses as well as the response times of a glucose biosensor have been studied under isothermal and non-isothermal conditions as a function of analyte concentration. The presence of a temperature gradient across the catalytic membrane system improved the biosensor characteristics, because the dynamic and steady-state responses increased and the response times decreased under non-isothermal conditions. For example, a macroscopic temperature difference of 20 degrees C applied across the catalytic membrane system increases the biosensor sensitivity of 70% and reduces of 50% its response time. The dependence of the observed effects on the magnitude of the temperature difference applied has been correlated with the substrate (and products) transport across the catalytic membrane system due to the process of thermodialysis.     

Independent control of respiration in cytochrome c oxidase vesicles by pH and electrical gradients

The effects of altering the pH and electrical components of the membrane potential on the visible spectra and oxygen consumption rates of cytochrome oxidase vesicles were examined during steady-state respiration using cytochrome c as the substrate. Heme a was found to be 30-55% reduced in the presence of a membrane potential, becoming more reduced when the electrical gradient (delta psi) was abolished by valinomycin and more oxidized when the pH gradient (delta pH) was abolished by nigericin, with little increase (1.2-1.8-fold) in the rates of oxygen consumption in either case. When both gradients were eliminated, heme a reduction was close to initial levels, and activity was stimulated up to 8-fold. The magnitude of the changes in heme a reduction levels upon elimination of a gradient component was shown to be positively correlated with the magnitude of the respiratory control ratio of the vesicle preparation. Kinetic analysis of the dependence of oxidase activity on cytochrome c concentration indicated that changes in the Michaelis constant of the enzyme for its substrate are not a major factor in regulation by either delta pH or delta psi. These results suggest a dual mechanism for respiratory control in cytochrome oxidase vesicles under steady-state conditions, in which the electrical gradient predominantly affects electron transfer from cytochrome c to heme a, possibly by altering the reduction potential of heme a, while the pH gradient affects electron transfer from heme a (CuA) to heme a3 (CuB), possibly by a conformationally mediated change in the reduction potential of heme a3 or in the kinetics of the electron-transfer process.     

Salt-induced absorbance changes of P-515 in broken chloroplasts

Absorbance changes, caused by adding KCl to a suspension of broken chloroplasts in the presence of a low concentration of MgCl2, have been measured in the wavelength region 460-540 nm. The magnitude of the KCl-induced absorbance changes is shown to be proportional to the logarithm of the KCL concentration gradient initially induced across the thylakoid membrane. The difference spectrum of these absorbance changes is shown to be identical with the spectrum of the light-induced absorbance changes, which has been attributed to an electrochromic shift of p-515. This is interpreted as evidence that under these conditions salt-induced absorbance changes of P-515 occur in response to a membrane diffusion potential. The results indicate that the electrogenic potential across the thylakoid membrane, generated by a single turnover light flash, is in the range between 15 and 35 mV.     

Kinetic measurements of electron transfer in coupled chromatophores from photosynthetic bacteria. A method of correction for the electrochromic effects

A quantitative study of the kinetics of electron transfer under coupled conditions in photosynthetic bacteria has so far been prevented by overlap of the electrochromic signals of carotenoids and bacteriochlorophyll with the absorbance changes of cytochromes and reaction centers. In this paper a method is presented by which the electrochromic contribution at any wavelength can be calculated from the electrochromic signal recorded at 505 nm, using a set of empirically determined polynomial functions. The electrochromic contribution to kinetic changes at any wavelength can then be subtracted to leave the true kinetics of the redox changes. The corrected redox changes of the reaction center measured at 542 and 605 nm mutually agree, thus providing an excellent test of self-consistency of the method. The corrected traces for reaction center and of cytochrome b-566 demonstrate large effects of the membrane potential on the rate and poise of electron transfer. It will be possible to study the interrelation between proton gradient and individual electron reactions under flash or steady-state illumination.     

Salt-induced absorbance changes of P-515 in broken chloroplasts

Absorbance changes, caused by adding KCl to a suspension of broken chloroplasts in the presence of a low concentration of MgCl₂, have been measured in the wavelength region 460–540 nm. The magnitude of the KCl-induced absorbance changes is shown to be proportional to the logarithm of the KCl concentration gradient initially induced across the thylakoid membrane. The difference spectrum of these absorbance changes is shown to be identical with the spectrum of the light-induced absorbance changes, which has been attributed to an electrochromic shift of P-515. This is interpreted as evidence that under these conditions salt-induced absorbance changes of P-515 occur in response to a membrane diffusion potential. The results indicate that the electrogenic potential across the thylakoid membrane, generated by a single turnover light flash, is in the range between 15 and 35 mV.     

Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1.

The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated. Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells. Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1. At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH). At higher pH values, inhibition was observed only with valinomycin, an ionophore which collapses the electrical potential across the membrane (delta psi). Under anaerobic conditions, no uptake of 4-CBA was observed unless an alternative electron acceptor was present. With nitrate as the terminal electron acceptor, 4-CBA was rapidly accumulated by the cells to a steady-state level, at which uptake of 4-CBA was balanced by excretion of 4-hydroxybenzoate. The mechanism of energy coupling to 4-CBA transport under anaerobic conditions was further examined by the imposition of an artificial delta psi, delta pH, or both. Uptake of 4-CBA was shown to be coupled to the proton motive force, suggesting a proton symport mechanism. Competition studies with various substrate analogs revealed a very narrow specificity of the 4-CBA uptake system. This is the first report of carrier-mediated transport of halogenated aromatic compounds in bacteria.     

Photoresponse of chlorophyll-containing bileaflet membranes and the effect of phycocyanin as extrinsic membrane protein

Summary Artificial bileaflet membranes were formed from extracts of chloroplasts. Gradients of a redox potential were created across the membranes by adding various concentrations of ceric-cerous ions, ferric-ferrous ions, and ascorbic acid to the aqueous solutions on either side of the membrane. When a membrane interposed between solutions of different redox potential was irradiated with light, a potential difference of up to 50 mV was recorded. Analysis of the photoresponse allowed its separation into two components: a photoelectromotive driving force dependent upon the redox potential gradient, and a photoconductive pathway dependent upon the amount of light absorbed by the membranes. There appeared to be a limit to the photocurrent that could be drawn from a membrane at a particular intensity of irradiation; i.e., it did not increase indefinitely with increase of the redox potential gradient. Conductance of the photoconductive pathway was independent of temperature. Phycocyanin added to the aqueous solution participated in the photoresponse in a unidirectional manner that suggested facilitation of electron transport from membrane to acceptors in the aqueous solution.     

Dependence of photosensitivity of bileaflet lipid membranes upon the chlorophyll and carotenoid content.

Bileaflet lipid membranes were formed from solutions containing lecithin, chlorophyll and carotene in various concentrations. If all the above components were present at sufficient concentrations the membranes were photosensitive; i.e., a photocurrent was produced if a redox potential gradient was present across the membranes. The presence of chlorophyll and carotene were essential for the photosensitivity of the membranes. Photoresponse could be elicited by illuminating the membrane with light which did not excite carotene. On the other hand, elimination of the part of the light spectrum which excites chlorophyll led to the abolition of the photoresponse. The findings of this study are consistent with the assumption that the excited chlorophyll chromophores allow electron exchange at the membrane-water interface while the presence of carotene allows electron movement across the "bulk" lipid membrane.     

Protonmotive force and photophosphorylation in single swollen thylakoid vesicles

Swollen vesicles generally 40 micron in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin-luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a delta pH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.     

Steady-state measurements of ΔpH and Δψ in Rhodospirillum rubrum chromatophores by two different methods. Comparison with phosphorylation potential

The pH gradient, ΔpH, and the membrane potential, Δψ, formed during light-induced electron transport in Rhodospirillum rubrum chromatophores were measured by two independent methods: (a) using specific electrodes to monitor light-dependent uptake of NH₄Cl and SCN^? at chromatophore concentrations of about 0.1 mg bacteriochlorophyll/ml and (b) using 9-aminoacridine and 8-anilinonaphthalenesulfonic acid as fluorescent probes for ΔpH and Δψ, respectively, at chromatophore concentrations of about 0.01 mg bacteriochlorophyll/ml. The light intensity was measured and set at a level which saturated the highest bacteriochlorophyll concentration used. The steady-state values obtained with each method under phosphorylating conditions were compared with the phosphorylation potential maintained by the chromatophores under identical conditions. The results indicate that under all conditions employed the ratio $\text{H}{}^{\mathrm{+}}\text{ATP}$ is greater than 2, and varies between 2.4 and 3.4 depending on the method used for estimation of the electrochemical proton gradient.     

Measurement of the mitochondrial membrane potential and pH gradient from the redox poise of the hemes of the bc1 complex

The redox potentials of the hemes of the mitochondrial bc(1) complex are dependent on the proton-motive force due to the energy transduction. This allows the membrane potential and pH gradient components to be calculated from the oxidation state of the hemes measured with multi-wavelength cell spectroscopy. Oxidation states were measured in living RAW 264.7 cells under varying electron flux and membrane potential obtained by a combination of oligomycin and titration with a proton ionophore. A stochastic model of bc(1) turnover was used to confirm that the membrane potential and redox potential of the ubiquinone pool could be measured from the redox poise of the b-hemes under physiological conditions assuming the redox couples are in equilibrium. The pH gradient was then calculated from the difference in redox potentials of cytochrome c and ubiquinone pool using the stochastic model to evaluate the ΔG of the bc(1) complex. The technique allows absolute quantification of the membrane potential, pH gradient, and proton-motive force without the need for genetic manipulation or exogenous compounds.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium.

The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.     

The membrane potential of intact Rhodospirillum rubrum cells in the absence of light-dependent and oxygen-linked electron transfer

In the absence of oxygen-linked and light-dependent electron transfer, the steady-state membrane potential of intact Rhodospirillum rubrum cells was usually between 65 and 75% of that of dark aerated cells, as indicated by the relative extent of the bacteriochlorophyll electrochromic changes that were induced by oxygen and by uncouplers. That potential was not due to residual levels of oxygen or light, because its value was not significantly altered by the presence of oxygen-trapping systems or by exhaustive gassing with Ar, and because it was also exhibited by a reaction-center-less mutant. The dark anaerobic potential was unaffected by 0.11 M K⁺; that seemed to exclude a diffusion potential generated by dissipation of a previously built K⁺ gradient. In contrast, it was largely abolished by 0.5 mM N,N′-dicyclohexylcarbodiimide, suggesting its dependence on ATP hydrolysis by the proton-translocating ATPase of the bacterial membrane. That was not expected because R. rubrum did not grow fermentatively under the conditions used. Low concentrations of protonophores were more effective in dissipating the anaerobic than the aerobic membrane potential. That observation indicated a lower activity of the electrogenic system responsible for the anaerobic potential. In consequence, the addition of uncouplers at low levels resulted in a marked enhancement of the membrane potential decrease which followed the transition between the aerobic and the anaerobic steady states.