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J Fischbarg

Publications and source records attributed to J Fischbarg.

At least 55 records · Page 3Linked to original sources
PubMed1996

Monomeric human red cell glucose transporter (Glut1) in non-ionic detergent solution and a semi-elliptical torus model for detergent binding to membrane proteins.

The self-association state of the human red cell glucose transporter (Glut1) in octaethylene glycol n-dodecyl ether (C12E8) and n-octyl beta-D-glucopyranoside (OG) solution was analyzed in the presence of reductant by gel filtration with light-scattering, refractivity and absorbance detection, and by ultracentrifugation. The C12E8-Glut1 complex was essentially monomeric, whereas OG-Glut1 also formed dimers and larger oligomers. C12E8-Glut1 retained substantial glucose transport activity even after depletion of endogenous lipids by gel filtration, as shown by reconstitution and transport measurements. Removal of endogenous lipids from OG-Glut1 abolished the activity unless phosphatidylcholine was included in the eluent. The binding of C12E8 and OG to Glut1 was determined by gel filtration with refractivity and absorbance detection or with radioactive tracer to be 1.86 +/- 0.07 and 1.84 +/- 0.09 g/g polypeptide, respectively. A structural model was proposed in which non-ionic detergent forms a semi-elliptical torus (SET) surrounding the transmembrane protein. The torus thickness was assumed to be equal to the radius (short half-axis) of a spherical (oblate ellipsoidal) free detergent micelle and the polar head groups of the detergent molecules were predicted to be situated just outside the hydrophobic surface of the protein. The experimental detergent binding values and those obtained from the SET model together confirmed that Glut1 was monomeric in C12E8 solution and provided constraints on the shape and size of the hydrophobic transmembrane region of Glut1 in alpha-helical and beta-barrel topology models.

Biological Transport↗
PubMed1996

Modeling facilitative sugar transporters: transitions between single and double ligand occupancy of multiconformational channel models explain anomalous kinetics.

The four-state simple carrier model (SCM) is employed to describe ligand translocation by diverse passive membrane transporters. However, its application to systems like facilitative sugar transporters (GLUTs) is controversial: unidirectional fluxes under zero-trans and equilibrium-exchange experimental conditions fit a SCM, but flux data from infinite-cis and infinite-trans experiments appear not to fit the same SCM. More complex kinetic models have been proposed to explain this "anomalous" behavior of GLUTs, but none of them accounts for all the experimental findings. We propose an alternative model in which GLUTs are channels subject to conformational transitions, and further assume that the results from zero-trans and equilibrium-exchange experiments as well as trans-effects corresponds to a single-occupancy channel regime, whereas the results from the infinite-cis and infinite-trans experiments correspond to a regime including higher channel occupancies. We test the plausibility of this hypothesis by studying a kinetic model of a two-site channel with two conformational states. In each state, the channel can bind the ligand from only one of the compartments. Under single-occupancy, for conditions corresponding to zero-trans and equilibrium-exchange experiments, the model behaves as a SCM capable of exhibiting trans-stimulations. For a regime including higher degrees of occupancy and infinite-cis and infinite-trans conditions, the same channel model can exhibit a behavior qualitatively similar to a SCM, albeit with kinetic parameters different from those for the single-occupancy regime. Numerical results obtained with our model are consistent with available experimental data on facilitative glucose transport across erythrocyte membranes. Hence, if GLUTs are multiconformational channels, their particular kinetic properties can result from transitions between single and double channel occupancies.

Carbohydrate Metabolism↗
PubMed1996

Sodium-glucose cotransporters display sodium- and phlorizin-dependent water permeability.

Expression of Na(+)-glucose cotransporters of the SGLT-1 type by Xenopus laevis oocytes increased the osmotic water permeability (Pf) of oocytes by a factor of 1.9-2.8, in the presence and in the absence of 5 mM extracellular glucose. The Pf increase was correlated with the amount of SGLT-1 cRNA injected. In oocytes expressing SGLT-1, either addition of phlorizin to the medium or the replacement of Na+ by choline inhibited the uptake of methyl-alpha-D-glucopyranoside, a specific substrate for SGLT-1, and returned oocyte Pf to its level in uninjected oocytes. Phlorizin inhibited the SGLT-1-attributable increase in Pf with an inhibition constant (Ki) of 6.1 microM, a value analogous to the Ki for phlorizin inhibition of sugar uptake. However, neither the presence of phlorizin nor the absence of extracellular Na+ significantly affected the increase in Pf elicited in oocytes expressing GLUT-1, a facilitative glucose transporter. These findings suggest that SGLT-1 forms a pore that allows the transmembrane passage of water and that water and glucose traverse the protein through this pore. The finding that removal of extracellular Na+ abolishes the increase in Pf attributable to SGLT-1 suggests that extracellular Na+ is required to maintain patency of this transporter's water-permeable transmembrane pore.

Animals↗
PubMed1996

Platelet activating factor inhibits fluid transport by corneal endothelium.

PURPOSE: Given reports of corneal edema after endothelial exposure to platelet activating factor (PAF), the authors have investigated whether PAF can affect the function of corneal endothelium in vitro. METHODS: The endothelial side of deepithelialized rabbit corneas was perfused with BSS+ and test agents: PAF, its inactive receptor ligand analog Lyso-PAF, and its antagonist BN52021. Stromal thickness was determined by specular microscopy. Translayer-specific electrical resistance (rho) was measured in cultured bovine corneal endothelial cells grown on permeable substrates at 36.5 degrees C. RESULTS: Control corneas perfused with BSS+ or with BSS+ containing Lyso-PAF swelled at a very slow rate (6.2 +/- 0.1, and 7.9 +/- 0.2 microns/hour, respectively). Corneas exposed to PAF swelled appreciably faster and at rates that were a saturable function of PAF (K(m), 2.1 microM); maximal rates of swelling were < 20 microns/hour, indicating no appreciable damage to intercellular junctions. BN52021 prevented PAF-induced swelling (Ki, 1.1 microM). PAF led also to a decrease in rho (from 42.8 +/- 1.4 to 24.5 +/- 0.6 omega cm2 in 1 hour; 46.8 +/- 1.5 to 38.3 +/- 1.4 omega cm2 in control layers; and 43.0 +/- 1.2 to 30.8 +/- 1.6 omega cm2 in layers exposed to PAF+BN52021). Such rho changes are consistent with swelling of intercellular spaces. CONCLUSIONS: Results suggest that PAF inhibits transendothelial fluid transport on binding to an endothelial cell receptor for it; continuous stimulation of a PAF-induced signaling cascade may lead to such inhibition. From these and other results, fluid transport might result from cascades activating sequentially basolateral and apical transporters or channels.

Animals↗
PubMed1995

Osmotic permeability in a molecular dynamics simulation of water transport through a single-occupancy pore.

The aim of this work is to determine plausible values for the rate constants of kinetic models representing water transport through narrow pores. We present here the results of molecular dynamics simulations of the movement of water molecules through a single-site hydrophilic pore. The system consists of a rectangular box of water molecules, some of which are positionally restrained so as to act as a membrane. This membrane separates two compartments where water molecules move freely; one of the positions in the membrane is initially vacant (the 'single-site pore'), but can be occupied by mobile molecules. To analyze the results, we represented the pore by a two-state kinetic diagram in which the vacant and occupied states are linked by transitions corresponding to the binding and release of water molecules. The mean occupancy and vacancy times directly yield the rate constants of binding and release, which in turn yield the osmotic water permeability coefficient per pore pf. We also compute the apparent activation energies delta E* for the rate constants and for pf. The pf value was (1.56 +/- 0.04).10(-11) cm3/s (at 307 K), which is much larger than those determined for CHIP28 and for gramicidin A (of about 10(-13) and 10(-14) cm3/s, respectively). These values were compared with those arising from a model of a symmetric single-file pore through which one-vacancy-mediated water transport takes place. The model yields an expression for pf as a function of the rate constants and of the number of molecular positions (n) in the file. When n = 1, this expression becomes the one corresponding to the single-site pore studied in our current simulation. Using the rate constants of binding and release derived from our simulation, the pf values are consistent with an occupancy value of 5-6 found for gramicidin A, and with occupancies of 4-7 that can be estimated for the single-file pore of a recently proposed model for CHIP28. delta E* for pf is 3.0 kcal/mol, a value similar to that determined for CHIP28. Hence, the system simulated here appears plausible and can be used to mimic some physical properties of water transport through biological pores.

Biological Transport↗
PubMed1995

Predictive evidence for a porin-type beta-barrel fold in CHIP28 and other members of the MIP family. A restricted-pore model common to water channels and facilitators.

Water channels are the subject of much current attention, as they may be central for cell functions in a host of tissues. We have analyzed the possible field of facilitators and water channels of the MIP family based on structural predictions, on findings about the topology of CHIP28, and on the biophysical characteristics of water channels. We developed predictions for the following proteins: MIP26, NOD26, GLP, BIB, gamma-TIP, FA-CHIP, CHIP28k, WCH-CD1, and CHIP28. We utilized Kyte Doolittle hydrophobicity, Eisenberg's amphiphilicity, Chou-Fasman-Prevelige propensities, and our own Union algorithm. We found that hydrophobic amphiphilic segments likely to be transmembrane were consistently shorter than required for alpha-helical segments, but of the correct length for beta-strands. Turn propensity was high at frequent intervals, consistent with transmembrane beta-strands. We propose that these proteins fold as porin-like 16-stranded antiparallel beta-barrels. In water channels, from the size of molecules excluded, an extramembrane loop(s) would enter the pore and restrict it to a bottleneck with a width 4 A < or = w < or = 5 A. A similar but more mobile loop(s) would act as gate and binding site for the facilitators of the MIP family.

Amino Acid Sequence↗
PubMed1995

Multifunctional transporter models: lessons from the transport of water, sugars, and ring compounds by GLUTs.

Facilitative glucose transporters (GLUTs) have recently been shown to be multifunctional, transporting substrates other than sugars, such as water and ring compounds as large as nitrobenzene-diazol-aminoglucose. Other membrane proteins, including transporters and cystic fibrosis transmembrane conductance regulator, have also revealed a finite permeability to water. We compare the alpha-helical and beta-barrel models for the structure of GLUTs, discuss recent evidence, and argue that a beta-barrel fold explains it better. We show a model for GLUTs consisting of a relatively rigid beta-barrel translocation unit ("channel") of diameter ample enough to allow permeation of the above substrates (approximately 20 A) but gated shut by mobile loops at both ends. Such gates would open only after aromatic interactions would lead to binding of the ring substrates for GLUTs; water would, however, traverse crevices in the closed gates. Using the insights gained from GLUTs, we propose that other transporters may share with GLUTs the motif of a beta-barrel channel and would be permeable to water due to the presence of such channels together with similarly behaving gates.

Animals↗
PubMed1994

Are most transporters and channels beta barrels?

Given the sequence of transporters or channels of unknown secondary structure, it is usual to predict their putative transmembrane regions as alpha-helical. However, recent evidence for a facilitative glucose transporter (GLUT1) appears inconsistent with such predictions, which has led us to propose an alternative folding model for GLUTs based on the 16-stranded antiparallel beta-barrel of porins. Here we apply the same predictive algorithms we used for GLUTs to several other membrane proteins. For some of them, a high-resolution structure has been derived (beta-barrels: Rhodobacter capsulatus and Escherichia coli porins; multihelical: colicin A, bacteriorhodopsin, and reaction center L chain); we use them to test the prediction procedures. The other proteins we analyze (GLUT1, CHIP28, acetylcholine receptor alpha subunit, lac permease, Na(+)-glucose cotransporter, shaker K+ channel, sarcoplasmic reticulum Ca(2+)-ATPase) are representative of classes of similar membrane proteins. As with GLUTs, we find that the predicted transmembrane segments of these proteins are consistently shorter than expected for transmembrane spanning alpha-helices, but are of the correct length and number for the proteins to fold instead as porin-like beta-barrels.

Algorithms↗
PubMed1994

Transport properties of single-file pores with two conformational states.

Complex facilitative membrane transporters of specific ligands may operate via inner channels subject to conformational transitions. To describe some properties of these systems, we introduce here a kinetic model of coupled transport of two species, L and w, through a two-conformational pore. The basic assumptions of the model are: a) single-file of, at most, n molecules inside the channel; b) each pore state is open to one of the compartments only; c) there is at most only one vacancy per pore; d) inside the channel, a molecule of L occupies the same positions as a molecule of w; and e) there is at most only one molecule of L per pore. We develop a general representation of the kinetic diagram of the model that is formally similar to the one used to describe one-vacancy transport through a one-conformational single-file pore. In many cases of biological importance, L could be a hydrophilic (ionic or nonionic) ligand and w could be water. The model also finds application to describe solute (w) transport under saturation conditions. In this latter case, L would be another solute, or a tracer of w. We derive steady-state expressions for the fluxes of L and w, and for the permeability coefficients. The main results obtained from the analysis of the model are the following. 1) Under the condition of equilibrium of w, the expression derived for the flux of L is formally indistinguishable from the one obtainable from a standard four-state model of ligand transport mediated by a two-conformational transporter. 2) When L is a tracer of w, we can derive an expression for the ratio between the main isotope and tracer permeability coefficients (Pw/Pd). We find that the near-equilibrium permeability ratio satisfies (n - 1) < or = (Pw/Pd)eq < or = n, a result previously derived for the one-conformational, single-file pore for the case that n > or = 2. 3) The kinetic model studied here represents a generalization of the carrier concept. In fact, for the case that n = 1 (corresponding to the classical single-occupancy carrier), the near-equilibrium permeability ratio satisfies 0 < or = (Pw/Pd)eq < or = 1, which is characteristic of a carrier performing exchange-diffusion.

Binding Sites↗
PubMed1994

The "independence principle" in the processes of water transport.

The processes of membrane transport exhibiting permeability coefficients depending on the species activities do not obey the "independence principle" and are assumed to take place by a mechanism of discrete nature, analyzable by a kinetic formalism. In this article, we study the dependence of the osmotic permeability coefficient on the water activities, from the steady-state analysis of a kinetic model of single-file water transport that simultaneously incorporates the vacancy-mediated and "knock-on" mechanisms into the state diagram. In particular, we study the relation between the near-equilibrium osmotic permeability (Pe) and the equilibrium water activity of the compartments (w). The analysis and numerical calculations performed for a simple case of the model show that, for values of the parameters consistent with experimental data, Pe exhibits only a small variation with w within the physiological range in the majority of the situations considered here. It is not possible to predict, from the study of these simple models, whether more complicated kinetic diagrams of water transport may be characterized by permeability coefficients with a more evident dependence on the water activities. Nevertheless, the results obtained here suggest that, for the case of physiological water pores, the analysis of the kinetic dependence of the permeability coefficients on the water activities may not yield evidence pointing to a discrete nature for the transport process.

Animals↗
PubMed1993

Evidence that facilitative glucose transporters may fold as beta-barrels.

A widely accepted model for the structure of the facilitative glucose transporters (GLUTs) predicts that they form 12 transmembrane alpha-helices and that the highly conserved sequence Ile-386-Ala-405 in GLUT1 is intracellular. We raised a polyclonal antibody against a synthetic peptide encompassing this conserved sequence and found that antibody treatment increased 2-deoxy-D-glucose (DOG) uptake in Xe-nopus oocytes expressing GLUT1, GLUT2, or GLUT4 only when applied to the extracellular side. This effect was dose dependent and was specifically blocked by competition with the peptide Ile-386-Ala-405; it was due to a decrease in the Km for the transport of DOG. To ascertain GLUT orientation, we raised anti-peptide antibodies against the last 21 and 25 C-terminal amino acids of GLUT1 and GLUT4, respectively, which were previously shown to be intracellular. These antibodies increased DOG uptake when injected into oocytes expressing GLUT1 and GLUT4, but not when added extracellularly. Prompted by the noted discrepancy, we found sequence similarity between GLUTs and porins, two of which are known from crystallography to form 16-stranded transmembrane antiparallel beta-barrels. Analysis of the hydrophobicity, amphiphilicity, and turn propensity of GLUT1 leads us to propose that GLUTs fold as porin-like transmembrane beta-barrels. This model is consistent with the results of the present antibody studies and also with previously published experimental evidence inconsistent with the 12-helix model.

Amino Acid Sequence↗
PubMed1993

Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid.

Although vitamin C is critical to human physiology, it is not clear how it is taken up into cells. The kinetics of cell and tissue accumulation of ascorbic acid in vitro indicate that the process is mediated by specific transporters at the cell membrane. Some experimental observations have linked the transport of ascorbic acid with hexose transport systems in mammalian cells, although no clear information is available regarding the specific role(s) of these transporters, if any, in this process. Here we use the Xenopus laevis oocyte expression system to show that the mammalian facilitative hexose transporters are efficient transporters of the oxidized form of vitamin C (dehydroascorbic acid). Two transport pathways, one with low affinity and one with high affinity for dehydroascorbic acid, were found in oocytes expressing the mammalian transporters, and these oocytes accumulated vitamin C against a concentration gradient when supplied with dehydroascorbic acid. We obtained similar results in experiments using normal human neutrophils. These observations indicate that mammalian facilitative hexose transporters are a physiologically significant pathway for the uptake and accumulation of vitamin C by cells, and suggest a mechanism for the accumulation of ascorbic acid against a concentration gradient.

Animals↗
PubMed1993

Postvitrectomy hypotonia: the role of the vitreous and retinochoroidal lesions.

The pathomechanisms of hypotonia after vitrectomy remain obscure. To examine the possible escape of intraocular fluid through the ocular wall, hydraulic fluid conductivity was measured across preparations of retina, pigmented epithelium, and choroid isolated from rabbit eyes 1, 3, 5, or 7 weeks after the production of laser or cryopexy lesions. The hydraulic conductivity measured in a modified Fischbarg-Bourguet chamber, was 0 in controls. At 1 and 3 weeks after transpupillary diode laser coagulation of the equatorial retina, hydraulic conductivity was measurable. However, the conductivity of these preparations returned to 0 by 5 weeks. After transscleral cryopexy, conductivity remained elevated for 7 weeks. Break-up of residual cortical vitreous with hyaluronidase increased the conductivity of "mature" cryopexy lesions to the values obtained 1 week after injury. These results suggest that there is escape of fluid across retinochoroidal lesions; fluid conductance typically decreases over time; an intact cortical gel can hinder conductance and seal the retinochoroidal leak. Thus, the risk factors for hypotonia appear to include extensive retinochoroidal lesions and the absence of a normal cortical vitreous in aphakia and/or after aggressive vitrectomy.

Animals↗
PubMed1993

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Role of facilitative glucose transporters in diffusional water permeability through J774 cells.

We have reported previously that in the presence of an osmotic gradient, facilitative glucose transporters (GLUTs) act as a transmembrane pathway for water flow. Here, we find evidence that they also allow water passage in the absence of an osmotic gradient. We applied the linear diffusion technique to measure the diffusional permeability (Pd) of tritiated water (3H-H2O) through plasma membranes of J774 murine macrophage-like cells. Untreated cells had a Pd of 30.9 +/- 1.8 microns/s; the inhibitors of facilitative glucose transport cytochalasin B (10 microM) and phloretin (20 microM) reduced that value to 15.3 +/- 1.8 (50%) and 11.0 +/- 0.7 (62%) microns/s, respectively. In contrast, no significant effect on Pd was observed in cells treated with dihydrocytochalasin B (Pd = 28.4 +/- 1.5 microns/s). PCMBS (3 mM) inhibited glucose uptake by greater than 95%, and 3H-H2O diffusion by approximately 30% (Pd = 22.9 +/- 1.5 microns/s). The combination of cytochalasin B plus pCMBS reduced Pd by about 87% (Pd = 3.9 +/- 0.3 microns/s). Moreover, 1 mM pCMBS did not affect the osmotic water permeability in Xenopus laevis oocytes expressing the brain/erythroid form of facilitative glucose transporters (GLUT1). These results indicate for the first time that about half of the total Pd of J774 cells may be accounted for by water passage across GLUTs. Hence, they highlight the multifunctional properties of these transporters serving as conduits for both water and glucose. Our results also suggest for the first time that pCMBS blocks glucose transport without affecting water permeation through GLUTs. Lastly, because pCMBS decreases the Pd of J774 cells, this suggests the presence in their plasma membranes of another protein(s) exhibiting water channel properties.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Effects of BSS and BSS+ irrigation solutions on rabbit corneal transendothelial electrical potential difference.

Measurements of electrical potential difference across isolated rabbit corneal endothelium (transendothelial electrical potential difference, TEPD) were used to evaluate the effect of two commonly used commercially prepared intraocular irrigation balanced salt solutions, BSS and BSS+, together with our own standard bicarbonate-containing control solution (BS). The highest and most durable TEPD values were obtained by using BSS+; TEPD began at 550 microV, peaked at 600 microV 1 h later, and then decreased slowly down to some 300 microV after about 8 h. Gassing BSS+ with an air/5% CO2 mixture was optimal; gassing it with air reduced the TEPD values slightly, by about 10%. On the other hand, perfusion with BSS gassed with air had a rapid and marked adverse effect; the TEPD, which began at some 500 microV, fell down precipitously to about 50 microV after 1 hour. In contrast, perfusion with BSS on the endothelial side with the addition of BS to the stromal side, both sides being gassed with an air/5% CO2 mixture, maintained the TEPD at levels near those obtained with BSS+. We attribute the adverse effect by BSS to the absence of bicarbonate in it. From these and past results, we conclude that BSS cannot be recommended for intraocular use as a properly balanced saline solution.

Animals↗
PubMed1993

Laser light-scattering system for studying cell volume regulation and membrane transport processes.

A simple and relatively inexpensive device utilizing laser light scattering for the study of volume regulatory behavior and membrane transport phenomena in cells cultured on or affixed to a rigid substrate is described in detail. Validation of the method is provided by study of cell types with known volume regulatory responses. The method we describe has numerous advantages over currently available techniques used to monitor cell volume changes. These advantages include 1) the ability to rapidly detect and quantify small cell volume changes on-line, 2) the ability to maintain natural cell morphology, cell surface contacts, and cell-to-cell interactions, 3) the ability to easily control solution temperature and gas and solute composition, and 4) the ability to perform multiple perturbations in a single experiment. The light-scattering system we describe can be modified to allow for simultaneous measurement of light-scattering signals and fluorescence emission from intracellular ion-sensitive probes and membrane potential dyes. In addition, our method may be useful for the study of apical and basolateral membrane transport processes in epithelial monolayer cell cultures.

Animals↗