Characterization of a fluorescence assay to monitor changes in the aqueous volume of lipid vesicles

The fluorescent compound, 4',5'-bis[N,N-bis(carboxymethyl)aminomethyl] fluorescein (calcein) has been characterized for use in lipid vesicle studies. Particularly useful is its reaction with Co2+, which results in fluorescence quenching. This is accompanied by about a 10-nm blue shift in the uv absorbance bands and a small reduction in the visible absorbance band. For vesicle studies, Co2+ may be combined with citrate, which does not significantly hinder calcein quenching by Co2+. It does augment the absorbance of the metal ion. No significant interaction of citrate X Co2+ with phosphatidylserine vesicles was observed. Zn2+ is capable of displacing Co2+ and restoring calcein fluorescence. Fluorescence quenching due to formation of the calcein X Co2+ complex can also be reversed with EDTA. Thus, calcein is the basis of some simple reactions which can be used to assay changes in the aqueous volume of lipid vesicles.     

Potential-dependent phase partitioning of fluorescent hydrophobic ions in phospholipid vesicles

Summary Fluorescent, dansyl derivatives of triphenylalkylphosphonium ions have been synthesized and exhibit fluorescence intensities in small sonicated phospholipid vesicles that are dependent upon transmembrane potentials. The voltage-dependent fluorescence changes are a result of changes in quantum yield that accompany a voltage-dependent phase partitioning of the probe. This phase partitioning is easily quantitated by calibrating the intensities of totally membrane-associated and aqueous probe. The voltage-dependence is well accounted for by a simple thermodynamic model and allows an estimation of potentials from fluorescence intensities in small vesicle systems.     

Relationship of the Donnan potential to the transmembrane pH gradient in tracheal apical membrane vesicles

Summary Experiments were performed to determine the factors which contribute to the transmembrane pH gradient (pH) and the potential gradient () in apical plasma membrane vesicles isolated from bovine tracheal epithelium. As indicated by the accumulation of¹⁴C-methylamine, the vesicles maintained a pH (inside acidic) which was dependent upon the external pH. The pH was also proportional to the ionic strength of the suspending medium, suggesting that the H⁺ distribution was dictated by a Donnan potential. Measurements of the distribution of⁸⁶Rb⁺ demonstrated an electrical potential gradient across the vesicle membrane, inside negative which was proportional to the medium ionic strength. pH changed in parallel with in response to a variety of imposed conditions. These results are compatible with the existence of a H⁺ conductance in the vesicle membrane. Thus the endogenous electrical and proton gradients may be manipulated and used as a general experimental tool to complement kinetic analysis in investigations of transport mechanism using isolated vesicle preparations.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

Relationship of transepithelial electrical potential to membrane potentials and conductance ratios in frog skin

Summary Previous studies in anuran epithelia have shown that, after clamping the transepithelial voltage in symmetrical sequences for 4–6 min there is near-constancy of the rate of active Na transport and the associated oxidative metabolism, with a near-linear potential dependence of both. Here we have investigated in frog skin the cellular electrophysiological events associated with voltage clamping (V_t=inside-outside potential). Increase and decrease ofV_t produced converse effects, related directly to the magnitude ofV_t.Hyperpolarization resulted in prompt decrease in inward transepithelial currentI_t and increase in fractional outer membrane resistancefR₀ (as evaluated from small transient voltage perturbations) and in outer membrane potentialV₀. Overshoot ofV₀ was followed by relaxation to a quasi-steady state in minutes. Changes infR₀ were progressive, with half times of some 1–5 sec. Changes in transepithelial slope conductanceg_t were more variable, usually preventing precise evaluation of the outer and inner cell membrane conductancesg₀ andg_i. Nevertheless, it was shown thatg₀ is related inversely toV_t andV₀. Presuming insensitivity ofV_i toV_t, the dependence ofg₀ onV₀ in the steady state much exceeds that predicted by the constant field equation. Apparent inconsistencies with earlier results of others may be attributable to differences in protocol and the complex dependence ofg₀ onV₀ and/or cellular current. In contrast to previous findings in tight epithelia at open circuit, differences inV_t were associated with substantial differences infR₀ and inner membrane potentialV_i. Hyperpolarization ofV_t over ranges commonly employed in studies of active transport and metabolism appears to increase significantly the electrochemical work per Na ion transported.     

Fluorescence depolarization studies of the phase transition in multilamellar phospholipid vesicles exposed to 1.0-GHz microwave radiation

The phase transition in multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles was studied during exposure to continuous wave 1.0-GHz microwave radiation. Fluorescence depolarization measurements using a lipid-seeking molecular probe, diphenylhexatriene (DPH). were performed as a function of temperature. Semilog plots of microviscosity versus temperature illustrate the phase transition which shows a 5°C shift when the vesicles are treated with chloroform as a positive control. No shift of the phase transition was found during exposure to microwave radiation at specific absorption rates between 1 and 30 W/kg. Samples were exposed in a rectangular transmission line (TEM cell), and specific absorption rates were calculated from electrical measurements of incident, reflected, and transmitted power. Samples were exposed to increasing intensities of radiation, while the temperature was maintained at either 23.5 or 25.5 °C; these temperatures represented the two ends of the phase transition region for these vesicles. No statistically significant difference was found between exposed and control samples. These results are in contrast to those of others using laser Raman spectroscopy to measure the phase transition in similar multilamellar vesicles exposed to microwave radiation.     

Transmembrane distribution of lipophilic cations in response to an electrochemical potential in reconstituted cytochromec oxidase vesicles and in vesicles exhibiting a potassium ion diffusion potential

It has been shown previously that biogenic amines and a number of pharmaceutical agents can redistribute across vesicle membranes in response to imposed potassium ion or proton gradients. Surprisingly, drug accumulation is observed for vesicles exhibiting either a pH gradient (interior acidic) or a membrane potential (interior negative), implying that these compounds can traverse the lipid bilayer as either the neutral or charged species. This interpretation, however, is complicated by the fact that vesicles exhibiting a membrane potential (interior negative) accumulate protons in response to this potential, thereby creating a pH gradient (interior acidic). This raises the possibility that in both vesicle systems drug redistribution occurs in response to the proton gradient present. We have therefore compared the uptake of several lipophilic cations by reconstituted cytochromec oxidase vesicles and by similar vesicles exhibiting a potassium ion diffusion potential. While turnover of the oxidase generates a membrane potential of comparable magnitude to the potassium ion diffusion system, it is associated with a proton gradient of opposite polarity (interior basic). Both systems show rapid uptake of the permanently charged lipophilic cation, tetraphenylphosphonium, but only the potassium ion diffusion system accumulates the lipophilic amines doxorubicin and propranolol. This provides compelling evidence that such weak bases redistribute only in response to pH gradients and not membrane potential.     

Binding of ferredoxin to ferredoxin:NADP+ oxidoreductase: the role of carboxyl groups, electrostatic surface potential, and molecular dipole moment.

The small, soluble, (2Fe-2S)-containing protein ferredoxin (Fd) mediates electron transfer from the chloroplast photosystem I to ferredoxin: NADP+ oxidoreductase (FNR), a flavoenzyme located on the stromal side of the thylakoid membrane. Ferredoxin and FNR form a 1:1 complex, which is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. We have used differential chemical modification of Fd to locate aspartic and glutamic acid residues at the intermolecular interface of the Fd:FNR complex (both proteins from spinach). Carboxyl groups of free and FNR-bound Fd were amidated with carbodiimide/2-aminoethane sulfonic acid (taurine). The differential reactivity of carboxyl groups was assessed by double isotope labeling. Residues protected in the Fd:FNR complex were D-26, E-29, E-30, D-34, D-65, and D-66. The protected residues belong to two domains of negative electrostatic surface potential on either side of the iron-sulfur cluster. The negative end of the molecular dipole moment vector of Fd (377 Debye) is close to the iron-sulfur cluster, in the center of the area demarcated by the protected carboxyl groups. The molecular dipole moment and the asymmetric surface potential may help to orient Fd in the reaction with FNR. In support, we find complementary domains of positive electrostatic potential on either side of the FAD redox center of FNR. The results allow a binding model for the Fd:FNR complex to be constructed.     

The Paramecium circadian clock: synchrony of changes in motility, membrane potential, cyclic AMP and cyclic GMP

The behavior of a ciliate protozoan, Paramecium, is known to represent the electrical state of the cell membrane, and regulation of the membrane potential and ciliary motion are known to involve cAMP and cGMP. The present study shows the synchrony of circadian changes in motility, resting membrane potential and cyclic nucleotides in P. multimicronucleatum. Using an automated system for tracking isolated single microorganisms, the isolated Paramecium cells are confirmed to swim fast and straight during the day (and subjective day) and slowly, with frequent turning, at night (and subjective night). The resting membrane potential is more negative during the day than at night. cAMP and cGMP concentrations oscillate in a manner, such that both cAMP and cGMP are higher during the day (or subjective day) than at night (or subjective night). The ratio of cGMP to cAMP during the light and dark cycle (LD) fluctuates, paralleling the fluctuation of the resting membrane potential measured during the LD. These results suggest that the Paramecium will provide an excellent model to explore daily and circadian orchestration of second messengers mediating signals from ambient light/dark cycles and circadian pacemaker to ion channels and cilia, directly involved in daily and circadian cellular outputs of resting membrane potential and motility. Accepted: 23 January 1997     

The kinetic mechanism of the glutamate-aspartate carrier in rat intestinal brush-border membrane vesicles: the role of potassium

The sodium dependent transport system for L-glutamate and L-aspartate localized in the apical part of rat enterocytes has previously been kinetically characterized (Prezioso, G., and Scalera, V. (1996). Biochim. Biophys. Acta 1279, 144–148). In this paper the mechanism by which the potassium cation specifically activates the L-glutamate–sodium cotransport process is investigated. Potassium has been found to act as an activator when it is present inside the membrane vesicles, while its presence outside is ineffective, and the effect is saturable. The kinetic parameters with respect to sodium and glutamate have been compared in the presence and in the absence of the activator. The results indicate that the ordered sodium–sodium glutamate mechanism is not altered by potassium, and that the activation is probably exerted on both the rate determining steps of the transport process. It is proposed that (1) a specific binding site for potassium is present on the inside hydrophilic part of the membrane carrier, (2) the binding of the effector accelerates the intramembrane rearrangement steps of both the disodium glutamate–carrier complex and the free carrier, (3) the affinity of the carrier is lowered with respect to sodium whereas it is increased for glutamate, and (4) K⁺ antiport is not performed by this carrier.     

Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function

The transport of mineral ions into and out of tissues and cells is central to the life of plants. Ion transport and the plasma membrane transporters themselves have been studied using a variety of techniques. In the last 15 years, measurement of specific ion fluxes has contributed to the characterization of transport systems. Progress in molecular genetics is allowing gene identification and controlled expression of transporter molecules. However the molecular expression of transporter gene products must be characterized at the functional level. The ion‐selective microelectrode technique to measure specific ion fluxes non‐invasively is ideally suited to this purpose. This technique, its theory, its links with others and its application and prospects in plant science, are discussed. Ions studied include hydrogen, potassium, sodium, ammonium, calcium, chloride and nitrate. Applications discussed include: solute ion uptake by roots; gravitropism and other processes in the root cap, meristematic and elongation zones; Nod factor effect on root hairs; osmotic and salt stresses; oscillations; the effects of light and temperature. Studies have included intact roots, leaf mesophyll and other tissues, protoplasts and bacterial biofilms. A multi‐ion capability of the technique will greatly assist functional genomics, particularly when coupled with imaging techniques, patch clamping and the use of suitable mutants.     

A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATPase in membrane vesicles

1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160–1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127–130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state pH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H⁺ transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca²⁺. When Ca²⁺ was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H⁺-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino] propane - DEAE diethylaminoethyl - EGTA ethylene glycolbis(-aminoethyl ether)-N,N,N,N,-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - PAF platelet-activating - factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine     

The citrus leaf cuticle in relation to measurement of leaf water potential using thermocouple psychrometers*

Abstract. Cuticular resistance to water vapour diffusion is an important aspect of thermocouple psychrometry and may introduce significant error in the measurement of leaf water potential (Ψ). The effect of the citrus (Citrus mitis Blanco) leaf cuticle on water vapour movement was studied using the times required for vapour pressure equilibration during thermocouple psychrometric measurement of Ψ. Cuticular abrasion with various carborundum powders was used to reduce the diffusive resistance of both the adaxial and abaxial leaf surfaces, and the extent of the disruption to the leaf was investigated with light and electron microscopy. Cuticular abrasion resulted in reduced equilibration times due to decreased cuticular resistance and greater water vapour movement between the leaf and the psychrometer chamber. Equilibration times were reduced from over 5 h in the unabraded control leaves to 1 h with cuticle abrasion. This was associated with the decrease in diffusive resistance with cuticular abrasion from over 55 s cm^?1 to less than 8 s cm^?1 for both the adaxial and abaxial leaf surfaces. Scanning electron micrographs of the abraded leaf tissue revealed considerable disruption of the stomatal ledge and of the guard cells, surface smoothing and displacement of waxes into the stomatal aperture, and damage to veins. Observations with the transmission electron microscope revealed frequent disruption of epidermal cell walls, and damage to both the cytoplasmic and vacuolar membranes.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Alternative hypotheses of proton ejection in cytochrome oxidase vesicles. Transmembrane proton pumping or redox-linked deprotonation of phospholipid-cytochrome c complex(es)

A review of published experimental and interpretative knowledge concerning proton ejection associated with cytochrome c oxidation by artificial phospholipid vesicles inlaid with cytochrome c oxidase indicates that the detailed characteristics of the redox-linked proton ejection cannot be simply explained by proton pumping. We propose an alternative hypothesis according to which proton ejection is due to the redox-linked deprotonation of a complex involving phospholipid and cytochrome c at the surface of the vesicles. The postulates upon which this hypothesis depends are explicitly outlined, and some methods of testing the hypothesis are suggested.     

Analysis of kinetic data in transport studies: New insights from kinetic studies of Na+-d-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling,rapid filtration apparatus

Summary Using the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991.J. Membrane Biol. 122:111–125), we have studied the kinetic characteristics of Na⁺-d-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35°C using a dynamic approach which involves linearregression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20°C, in the presence of high substrate concentrations or 1mm phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classicalv versus S plot. All together, our results support the existence of a single Na⁺-d-glucose cotransport system in these membranes for which Na⁺ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989.Am J. Physiol. 256:8618–8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters ofd-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved; (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.     

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: Role of Ca2+-dependent K+ channels

Summary Eleven different secosteroids or steroids (10^–10 to 10^–8 m) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibianNecturus kidney while recording basolateral cell membrane potentialV _m. Seven of these molecules (1,25(OH)₂D₃, 25(OH)D₃, 24,25(OH)₂D₃, 5,6-trans-25(OH)D₃, 19-diol-cholesterol, estradiol and testosterone) resulted in changes ofV _m (V _m) occurring in a few seconds, the largest V _m being observed with 1,25(OH)₂D₃, +6.5±0.75 mV (n=19); these seven (seco)steroids but not the four inactive sterols (vitamin D₃, cholesterol, 1D₃ and aldosterone) possess a hydroxyl group on at least one carbon of the C₁₇ to C₂₅ lateral chain of the sterol ring. The V _m effect was present in Na⁺-free or Cl^–-free media, but it was abolished in HCO₃-free media. Depolarization of cell membrane potential by addition of glucose, 11mm, in luminal perfusion fluid abolished the 1,25(OH)₂D₃-evoked V _m effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K⁺ conductances, suppressed the 1,25(OH)₂D₃-evoked V _m effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca²⁺-dependent K⁺ channels also abolished the 1,25(OH)₂D₃-evoked depolarization. Such observations are consistent with the presence of Ca²⁺-dependent K⁺ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)₂D₃ and probably by other (seco)steroids.     

The reproductive potential of Heterakis gallinarum in various species of galliform birds: implications for survival of H. gallinarum and Histomonas meleagridis to recent times

The reproductive potential of Heterakis gallinarum was substantially higher in the ring-necked pheasant than in any of the eight other species of galliform birds used on the 67 tests here reported. Pheasants on four tests yielded an average 19·4 times as many eggs that embryonated as were used to infect the birds, while for those on tests with a highly virulent strain of Histomonas meleagridis present the return was 21·1 eggs per egg used. Corresponding returns for chickens were 5·2 and 2·4; for guinea fowl, 9·7 and 1·3; and for turkeys, 1·9 and 0·17. Birds of the other five species gave even poorer returns. Previous studies had indicated that 10–30 times as many heterakid eggs must embryonate as survive to be ingested, under natural conditions. Inasmuch as the traditional host of Heterakis gallinarum must also have been that of the virulent strains of Histomonas meleagridis that have become man's contemporaries, we regard the ring-necked pheasant, or some very close relative, as being the most likely host of these parasites in the late Cenozoic and Recent Eras.     

The frequency of ion-pair substructures in proteins is quantitatively related to electrostatic potential: a statistical model for nonbonded interactions

A statistical analysis of ion pairs in protein crystal structures shows that their abundance with respect to uncharged controls is accurately predicted by a Boltzmann-like function of electrostatic potential. It appears that the mechanisms of protein folding and/or evolution combine to produce a "thermal" distribution of local nonbonded interactions, as has been suggested by statistical-mechanical theories. Using this relationship, we develop a maximum likelihood methodology for estimation of apparent energetic parameters from the data base of known structures, and we derive electrostatic potential functions that lead to optimal agreement of observed and predicted ion-pair frequencies. These are similar to potentials of mean force derived from electrostatic theory, but departure from Coulombic behavior is less than has been suggested.