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.     

The ferric-reducing activity of duodenal brush-border membrane vesicles is associated with a b-type haem

Rabbit brush-border membrane vesicles possess ferricyanide reducing activity. This activity is preferentially dependent on NADH as reductant, and can be stimulated by the addition of FMN. The latency of activity observed following vesicle solubilisation suggests that the responsible component is transmembranous, and partially sequestered on the inner-face of the vesicles prior to full solubilisation. Subsequent increases in detergent concentration (>0.3% w/v lauryl maltoside) were found to be inhibitory. Ferricyanide reducing activity was effectively inhibited by the sulphydryl modifying reagents N-ethyl malemide and p-chloromercuribenzoate, but not by the flavin analogue diphenylene iodonium. The ferric-reducing activity co-purified with a b-type haem when applied to Sephacryl S-200 columns. The putative cytochrome was found to be immunologically distinct from neutrophil cytochrome b₅₅₈     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Osmotic water permeability of small intestinal brush-border membranes

Summary A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90° to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25°C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43±0.044 sec^–1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25°C with an activation energy of 9.75±1.04 kcal/mole from 11 to 25°C and 17.2±0.55 kcal/mole from 25 to 37°C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51±0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 m radius, an osmotic permeability coefficient of 1.2×10^–3 cm/sec can be estimated for the native brush-border membranes at 25°C. These fesults are consistent with the solubility-diffusion model for water flow across small intestinal BBMV but are inconsistent with the existence there of large aqueous pores.     

Sodium uptake mechanisms in brush-border membrane vesicles prepared from rabbit renal cortex

Amiloride-sensitive and amiloride-insensitive components of ²²Na⁺ uptake were examined in brush-border membrane vesicles prepared from rabbit renal cortex. Both components could be stimulated by interior-negative electrical potentials, demonstrating a sodium conductance pathway and an effect of electrical potential on the initial rate of Na⁺/H⁺ exchange.     

Ion permeability of rabbit intestinal brush border membrane vesicles

Summary The ion permeability of rabbit jejunal brush border membrane vesicles was studied by measuring unidirectional fluxes with radioactive tracers and bi-ionic diffusion potentials with the potential-sensitive fluorescent dye, diS–C₃-(5). Tracer measurements provide estimates of the absolute magnitudes of permeability coefficients, while fluorescence measurements provide estimates of relative and absolute ion permeabilities. The magnitudes of the permeability coefficients for Na⁺, K⁺, Rb⁺, and Br^– were approximately 5 nanoliters/(mg protein × sec) or 10^–5 cm/sec as determined by radioactive tracer measurements. The apparent selectivity sequence, relative to Na⁺, as determined by bi-ionic potential measurements was: F^–, isetheionate, gluconate, choline (<0.1)+(1.0)–(1.5)=NO ₃ ^– (1.5)–(2.3)+(2.4)+(2.5)+(2.6)+(3.9) 4 ^– +(12)–(40). The origin of this selectivity sequence and its relationship to the ion permeability of the brush border membrane in the intact epithelium are discussed.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (β-alanyl-l-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na⁺, K⁺ or choline⁺ gradient conditions (extravesicular > intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na⁺-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a K_m of 9.6 ± 1.4 mM and a V_max of 2.9 ± 0.2 nmol / mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-l-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na⁺-independent process.     

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.     

H+/glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the pH-sensitive dye Acridine orange

Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H⁺/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.     

H+/glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the pH-sensitive dye Acridine orange.

Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H+/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.     

Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

Peptide transport in rabbit intestinal brush-border membrane vesicles studied with a potential-sensitive dye

Peptide transport in purified rabbit intestinal brush-border membrane vesicles has been studied using a potential-sensitive fluorescent dye, di-S-C3(5). Transport of dipeptides is accompanied by an increase in the fluorescence of the dye in the presence and absence of Na+, indicating electrogenic, Na+-independent peptide transport. Dipeptides containing D-amino acids also increase the fluorescence, showing that these peptides too possess significant affinity for the peptide transport system. beta-Alanylglycylglycine and prolylglycylglycine, very much like the dipeptides, increase the fluorescence even in the absence of Na+ which demonstrates the Na+-independent, electrogenic transport of tripeptides. However, concentrations needed for half-maximal fluorescence changes are higher for tripeptides than for dipeptides suggesting different affinities for the carriers. The studies, in addition, provide evidence for the existence of more than one carrier system for translocation of small peptides in rabbit intestinal brush-border membrane.     

Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.     

Critical carboxyl group(s) in Na-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na⁺ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na⁺-dependent transport systems, but not of the Na⁺-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na⁺ was observed, the parallelism between Na⁺-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na⁺/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

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.     

Na+-coupled glycine transport in reticulocyte vesicles of distinct sidedness: Stoichiometry and symmetry

Summary Sodium-coupled glycine transport has been studied using membrane vesicles of distinct sidedness, either inside-out or right side-out, prepared from sheep reticulocytes. The activity is chloride dependent and characterized by high and low apparent affinities for glycine (K _m 0.5mm and >10mm) for both types of vesicles as well as intact cells. Transport is symmetrical with respect to similar apparent affinity constants for glycine, for both the high- and low-affinity systems, and for sodium. Direct measurements of the sodium/glycine coupling indicate a ratio of 21, consistent with kinetic data fitted to a Hill-type equation describing glycine flux as a function of sodium concentration.     

Ion permeability of maize root membrane vesicles: Studies with light scattering

A possible modulation of permeabilities of membrane vesicles to anions and cations was explored by light scattering techniques, evaluated by measuring the capacity of the vesicles to shrink and swell in response to changes of the osmolarity of the incubation medium. Membrane fractions were obtained by phase partition. Purity was evaluated by detection and quantification of membrane enzyme markers: vanadate-sensitive ATPase for the plasma membrane, nitrate-sensitive ATPase for the tonoplast and azide-sensitive ATPase for mitochondria. Membrane vesicles (250 g protein) were exposed to hypertonic solutions of salts (0.6 osmolar). Kinetics of the changes in apparent absorbance at 546 nm were observed by the addition of potassium, nitrate and chloride salts. The diffusion of ions into vesicles was induced by an osmotic gradient across the membrane and brought about volume changes of vesicles. Upon addition of vesicles to the different solutions the following ion permselectivity sequences were observed: PNO ₃ ^– >PCl ^–>PSO ₄ ^2– and PK ⁺PNa ⁺>PNH ₄ ⁺.Abbreviations ATP adenosine 5-triphosphate - EDTA ethylene diaminetetraacetic acid - Tris-Mes (Tris[hydroxymethyl]aminomethane, Mes-(2-[N-Morpholino]ethanesulfonic acid) - PEG polyethylene glycol     

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.