Carrier Membrane Research Articles

Water Flux across Neutral Carrier Membranes

We investigated the water flux through neutral carrier membranes caused by the osmotic pressure difference of the solutions on both sides of these membranes. Miniaturized thin-film ion-selective electrodes were used as their extremely small internal electrolyte volume as well as their extremely thin membrane make them specifically suitable for such studies. The dilution of the internal electrolyte due to the transmembrane water flux results in a significant shift of the ISE potentials. A model explaining the potential versus time curve of thin-film sensors was developed. The penetration coefficient of water is determined experimentally with respect to the lipophilicity of the membrane plasticizer used. The inverse of the square root of the penetration coefficient depends linearly on the logarithm of the lipophilicity for almost all investigated membranes.

Tyrosine phosphorylation of selected secretory carrier membrane proteins, SCAMP1 and SCAMP3, and association with the EGF receptor.

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in "patches" at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP-EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

Structural and functional analysis of the porcine secretory carrier membrane protein 1 gene (SCAMP1).

The secretory carrier membrane proteins (SCAMPs) are highly conserved integral vesicle membrane components of the post-Golgi secretory and endocytic pathways. We have isolated and characterized the porcine SCAMP1 cDNA and gene coding for a variant of the SCAMP family. The SCAMP1 cDNA has a length of 3827 bp including a 133-bp 5' and 2701-bp 3' untranslated region. The mRNA has an open reading frame of 1014 nt coding for a protein of 338 amino acids with a calculated molecular mass of 37.9 kDa and a pI of 7.9. The porcine SCAMP1 is 97.04% identical with the human and rat paralogs, respectively. The SCAMP1 gene consists of nine exons with sizes ranging from 78 to 2842 bp and spans at least 70 kb of genomic DNA on porcine Chromosome (Chr) 2q21-q22. The promoter of the SCAMP1 gene is TATA-box-less, and transcription starts at a G-nucleotide 133 nt upstream the start codon.

UV-visible spectroscopic study of the reaction kinetics of methylpiperazine-modified poly(vinyl chloride)s for use as fixed-state proton carrier membranes

Aminated poly(vinyl chloride)s (AmPVCs) were synthesized for use as fixed-site proton carrier membranes. Reactions between PVC and methylpiperazine (MePIP) in dimethylformamide (DMF) were studied using UV-VIS spectroscopy over time, temperature, and in the presence of the catalyst, potassium fluoride (KF). Based on the spectra of AmPVCs in THF, the peak attributions were assigned and the extinction coefficients were calculated to determine the vinyl MePIP groups and polyenes with different number of conjugated double bonds ( n). The rate constants for the accumulation of vinyl MePIP groups ( k N ), polyenes with 2≤ n≤7 ( k n ), and total conjugated double bonds ( k D ) were calculated at 90°C. The k N constant was only about one half as much as k D , and k n > k n+1 (for 2≤ n≤7). Assuming an Arrhenius relationship, the activation energies (Δ E) were calculated for the formation of the vinyl MePIP groups and polyenes with 2≤ n≤13 over different temperature ranges and in the presence or absence of KF. The first double bond formation was always favoured over the substitution reaction at any temperature and were independent of the presence of KF. As for the propagation of double bonds, short chains grew easier than long chains; but after a certain n, they grew with the same energy barrier. When the EMF-pH responses of AmPVC membranes were studied, no clear dependence was observed between the Nernstian responses of the membranes and the amount of conjugated double bonds ( D) or the D/ R N , where R N was the amount of vinyl MePIP groups. Nernstian responses were obtained for membranes made from AmPVCs with 0.003≤ R N ≤0.04 and 0.6≤ D/ R N ≤1.7.

Cloning and analysis ofpsam2, a gene fromPisum sativumL. regulated in symbiotic arbuscular mycorrhiza and pathogenic root–fungus interactions

A differential RNA display fragment was used to isolate a full size clone from a cDNA library prepared fromPisum sativumL. root RNA. The corresponding gene,psam2, belongs to a gene family and its RNA accumulation was repressed in roots after inoculation with the arbuscular mycorrhizal fungusGlomus mosseae. No significant changes in transcript levels could be observed in the nodule symbiosis formed withRhizobium. RNA accumulation was, however, enhanced after infection with two fungal root pathogens,Chalara elegansandAphanomyces euteiches.Phosphate or nitrate fertilization had no influence onpsam2RNA expression. Sequencing revealed that the putative gene product is similar to animal secretory carrier membrane proteins. Its possible involvement in changes in root exuded compounds after mycorrhizal formation or in secretion processes in phytopathogenic interactions is discussed.

Influence of potassium fluoride on the syntheses of methylpiperazine-modified poly(vinyl chloride)s for use as fixed-site proton carrier membranes.

Aminated poly(vinyl chloride) (PVC) membranes were prepared that had a Nernstian response over a wide range of pH. The reaction between PVC and methyl-piperazine (MePIP) in dimethylformamide (DMF) was studied over a wide range of time and temperature, and in the presence of the catalyst, potassium fluoride (KF). Time, temperature, and KF increased the nitrogen (N) content of the resulting polymers, but sometimes at the expense of decreasing their specific viscosities (molecular weights). Activation energies of processes that occurred in different temperature ranges were estimated assuming an Arrhenius relationship. A Nernstian response occurred once the N content approached to about 0.3 w/w %, and was accelerated by the presence of KF at elevated temperatures. Increasing the N content above about 3% led to a loss of the Nernstian response either because of an increase in the double bond content and a subsequent decrease in polymer mobility, or because of a decrease in the molecular weight of the copolymer and concomitant difficulties in film preparation.

Two distinct populations of synaptic-like vesicles from rat brain.

In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain.

A genetic defect of an iron pump on chromosome 20 is postulated to cause human hemochromatosis

Hemochromatosis is postulated to be caused by a heterozygous or homozygous defect of a gene on the short arm of the human chromosome 20 coding for an ubiquitous iron carrier membrane protein responsible for transporting iron ions out of cells. This iron pump would be very important to stabilize the membrane potential and to avoid excessive intracellular storage of iron with the consequence of damage to cells by lipid peroxidation of membranes.

Three mammalian SCAMPs (secretory carrier membrane proteins) are highly related products of distinct genes having similar subcellular distributions.

The primary structures of three human forms of secretory carrier membrane proteins (SCAMPs) have been deduced from full-length clones isolated from a HeLa cell cDNA library and confirmed by a combination of comparison to expressed sequence tags, microsequencing of purified protein, and in vitro transcription and translation. The structures indicated that SCAMPs are highly related products of distinct genes, and that the sequence identity of an individual SCAMP between different mammalian species is almost complete. Analysis of the distribution of SCAMPs among different mammalian tissues and cells indicates parallel expression of polypeptides and cognate mRNAs, and indicates that the three SCAMPs are usually but not always expressed together. The apparent M(r)s of two SCAMPs (1 and 2) do not vary appreciably among species, while that of the third (SCAMP3) is approximately 2 kDa larger in rodent cells than in humans. Examination of the codistribution of the three forms within individual cells using double label immunofluorescence indicates extensive colocalization of SCAMP2 and SCAMP3 with endogenous SCAMP1, however, subcellular regions enriched for a particular SCAMP are readily visible. These findings suggest that the SCAMPs may largely function at the same sites during vesicular transport rather than in separate post-Golgi recycling pathways.

Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles.

The tubulovesicles of gastric parietal cells sequester H+/K+-ATPase molecules within resting parietal cells. Stimulation of parietal cell secretion elicits delivery of intracellular H+/K+-ATPase to the apically oriented secretory canaliculus. Previous investigations have suggested that this process requires the regulated fusion of intracellular tubulovesicles with the canalicular target membrane. We have sought to investigate the presence of critical putative regulators of vesicle fusion on immunoisolated gastric parietal cell tubulovesicles. Highly purified tubulovesicles were prepared by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the alpha subunit of H+/K+-ATPase. Western blot analysis revealed the presence of Rab11, Rab25, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs) on immunoisolated vesicles. The same cohort of proteins was recovered on vesicles immunoisolated with monoclonal antibodies against SCAMPs and VAMP-2. In contrast, whereas immunoreactivities for syntaxin 1A/1B and synaptosome-associated protein (SNAP-25) were present in gradient-isolated vesicles, none of the immunoreactivity was associated with immunoisolated vesicles. The observation of VAMP-2 and two Rab proteins on immunoisolated H+/K+-ATPase-containing tubulovesicles supports the role for tubulovesicles in a regulated vesicle fusion process. In addition, the presence of SCAMPs along with Rab11 and Rab25 implicates the tubulovesicles as a critical apical recycling vesicle population.

Evidence for colocalization and interaction between 37 and 39 kDa isoforms of secretory carrier membrane proteins (SCAMPs).

Secretory carrier membrane proteins (SCAMPs) are proteins of post-Golgi recycling carriers, including regulated secretory organelles. The two major size variants, SCAMP1 (37 kDa) and SCAMP2 (39 kDa), extensively colocalize in membranes of fibroblasts and parotid acinar cells based on immunocytochemistry and velocity centrifugation, although the relative amounts of each variant may differ in selected organelles. SCAMP1, and to a lesser extent, SCAMP2, are substrates for chemical crosslinking in situ, and the recognizable crosslinking products of SCAMP1 suggest potential formation of homomultimers. SCAMP1 and SCAMP2 can be co-immunoprecipitated following detergent solubilization, using antibodies that specifically react with only one of the variants. Both the localization and interactions of SCAMPs are reiterated using transfected SCAMP1 that is epitope tagged (myc) at either the NH2 or COOH terminus and an anti-myc antibody. Like other transport vesicle membrane proteins, SCAMPs form complexes that apparently include homomultimers. Furthermore, these studies suggest that both SCAMP1 and SCAMP2 may function together in a single protein complex.

Transport of ionic species through functionalized poly(vinylbenzyl chloride) membranes

Solid polymeric membranes of poly(vinylbenzyl chloride) (VBC), lightly crosslinked with divinyl benzene, were incompletely reacted such that a fraction of the benzyl chlorines in different membranes was replaced with either dimethyl phosphonate esters (MPE) or triethyl ammonium chloride groups (QA). This work was conducted in an effort to investigate ionic transport through charged and uncharged membranes and to develop fixed site carrier membranes to facilitate the transport of selected metal ions from an aqueous feed stream to a concentrated acid receiving stream. Bulk solution flow does not occur through these membranes. Instead, solute diffusion occurs through the membrane matrix. The effects of hydrogen ion gradient, metal ion identity and charge, reactive site type, acid type, and ionic strength on metal ion transport were investigated. Results are also presented on the effect of membrane orientation and pretreatment (swelling) on metal ion transport. Facilitated transport was investigated through the testing of membranes with varying MPE percent functionalization. The results presented compare the metal ion transport rate to the functionalization of the membranes.

Insulin-induced Recruitment of Glucose Transporter 4 (GLUT4) and GLUT1 in Isolated Rat Cardiac Myocytes: EVIDENCE OF THE EXISTENCE OF DIFFERENT INTRACELLULAR GLUT4 VESICLE POPULATIONS

Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane proteins (SCAMPs) in intracellular membranes. Insulin induced 5.7- and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the nonpermeant photoaffinity label [3H]2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis-(D-mannos-4-yloxy)propyl-2-amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction ( approximately 30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti-GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different immunoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracellular GLUT4 membranes with 1F8-acrylamide. Acute insulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes.

Ultramicroelectrodes for membrane research

This article describes the construction, testing, calibration and application of ion-selective double-barrelled microelectrodes based on neutral carrier or ion-exchanger liquid membranes. At present the use of ion-selective microelectrodes is the only method available for the direct and continuous measurement of ion activities in the intra- and extracellular spaces, on membrane surfaces, and even in subcellular organelles of a variety of intact cells. Intracellular ion activities in identified neurones of the central nervous system of the leech ( Hirudo medicinalis L.) have been measured with K +-, Na +-, H +-, Mg 2+- and Ca 2+-selective double-barrelled microelectrodes under various experimental conditions, such as changes in ion composition of the external medium, application of neurotransmitters and exposure to drugs. This approach has provided significant information about electrochemical potentials of ions, properties of receptor-coupled ion channels, and membrane transport related to ion activities in this nervous system.

High permeation of L-type Ca2+ channels at physiological [Ca2+]: homogeneity and dependence on the alpha 1-subunit.

Molecular cloning has identified multiple isoforms of dihydropyridine-sensitive C-class L-type Ca2+ channels. We tested the hypotheses that L-type (C-class) channels exhibit homogeneous high permeation properties at physiological Ca2+ concentrations and membrane potentials. We measured unitary currents through single dihydropyridine-sensitive omega-conotoxin-insensitive endocrine and smooth muscle L-type Ca2+ channels in rat pituitary GH3 and rat aortic A7r5 cell lines. We also measured unitary currents through smooth muscle (Cb) Ca2+ channel alpha 1-subunits in Chinese hamster ovary (CHO) cells. Our results show that single channel conductances of all three L-type (C-class) channels are uniform with high Ba2+ concentrations, e.g., approximately 23 pS with 110 mM Ba2+. The single channel conductances were reduced to similar values when the Ba2+ concentration was lowered to near-physiological values: 11.1, 9.3, and 8.4 pS in GH3, A7r5, and CHO cells at 2 mM Ba2+, respectively. The single channel conductances were not significantly different with near-physiological Ca2+ concentrations: 5.5, 5.9, and 4.9 pS in GH3, A7r5, and CHO cells at 2 mM Ca2+, respectively. The data suggest that L-type (C-class) channels are homogeneous in terms of Ca2+ permeation at physiological charge carrier concentrations and membrane potentials. Furthermore, the data indicate that the relatively high Ca2+ permeation under physiological conditions is determined by the intrinsic properties of the pore-forming Ca2+ channel alpha 1-subunit.

Olefin separation using silver impregnated ion-exchange membranes and silver salt/polymer blend membranes

Olefin separations through silver containing carrier membranes were investigated. A AgBF 4/Nafion blend membrane and Nafion 117 membrane exchanged with Ag + were used. A dry AgBF 4/Nafion blend membrane showed butene selectivity over n-butane with dry feed butene pressures below the dissociation pressure. This result is evidence that there was some hydration of the Ag + so the complexation was a gas-liquid type reaction. Moreover, the selectivity decreased with time, and after eight days of operation, the butene flux approached the n-butane flux even though the butene feed pressure was above the dissociation pressure. The AgBF 4/Nafion blend membrane showed a high facilitation effect for C 4 separation with low humidity in comparison to Nafion ion-exchange membranes which require high humidity. A 1-butene flux of 8 × 10 −8 mol/cm 2 s and 1-butene selectivity of 50 over n-butane was measured with the AgBF 4/Nafion blend membrane from a 1-butene n- butane 80 20 % feed mixture at 86 kPa total presure with only 20% relative humidity at 50°C. The membrane showed a 1,3-butadiene selectivity of 2 over 1-butene, and the total flux was greatly reduced by the presence of 1,3-butadiene. C 6 unsaturated hydrocarbon transport through these membranes was also investigated. Both AgBF 4/Nafion blend membranes and Nafion ion-exchange membranes showed selectivity for 1,5-hexadiene over 1-hexene. The Nafion ion-exchange membrane showed better performance for the C 6 separation. Heat treated Nafion 117 containing silver ions produced a higher separation factor and flux than obtained with a normal silver form Nafion 117 membrane.

Selective permeation of CO 2 through poly 2-( N, N-dimethyl)aminoethyl methacrylate membrane prepared by plasma-graft polymerization technique

A membrane having an amine moiety was prepared by plasma-grafting 2-( N, N-dimethyl)aminoethyl methacrylate (DAMA) onto a microporous polyethylene substrate. Permselectivity of the membrane for CO 2 over N 2 was achieved in both dry and water swollen conditions. When the CO 2 partial pressure in the feed gas was 0.047 atm, the selectivity of CO 2 over N 2 reached 130 for the highly swollen water containing membrane. This value was found to agree with that obtained with a mobile carrier membrane (supported liquid membrane) using DAMA as the carrier. The effects of several experimental conditions such as degree of grafting, feed partial pressure and temperature on the membrane performance were studied. It was suggested that the membrane acted as a fixed carrier membrane for CO 2 facilitated transport in under the dry condition and acted as a fixed reaction site membrane in the water swollen condition. The carrier transport mechanism is discussed for dry and aqueous membranes.

Glut4 Is Targeted to Specific Vesicles in Adipocytes of Transgenic Mice Overexpressing Glut4 Selectively in Adipose Tissue

Adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue (Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4-containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulin-sensitive vesicles as in normal cells.

Hybrid liquid membrane (HLM) system in separation technologies

A novel liquid membrane system, denoted hybrid liquid membrane (HLM), was developed for the separation of solutes (metal ions, acids, etc.). It utilizes a solution of an extracting reagent (carrier solution), flowing between membranes. The membranes, which separate the carrier solution from feed and receiving (strip) solutions, enable the transport of solutes, but block the transfer of the carrier to the feed or to the strip. Blocking the carrier is achieved through membranes hydrophilic/hydrophobic or ion exchange properties, or through their rentention abilities, due to pore size. The HLM-facilitated transport mechanisms have been schematically described and theoretical models have been developed to predict the rate of transport in the different separation processes. The model was tested on titanium(IV) transport from hydrochloric acid solutions. Titanium was removed by counter-transport from low acidic (pH = 0.65) solutions or by co-transport from high acidic (7 mol/kg HCl) solutions, using DEHPA in benzene as a liquid carrier (membrane) solution. The efficiency of titanium transfer was studied as a function of feed, carrier and strip flow rate. Mass transfer parameters obtained were compared with model calculated data. Module optimization characteristics are discussed.

Analysis of facilitated transport in solid membranes with fixed site carriers 1. Single RC circuit model

A simple mathematical model for facilitated mass transport with a fixed site carrier membrane was derived by assuming an instantaneous, microscopic concentration (activity) fluctuation in the membrane. The concentration fluctuation, developed due to the reversible chemical reaction between carrier and solute, could cause the higher chemical potential gradient and the facilitated transport. For mathematical formulation, the fluctuated concentration profile was assumed to be a sinusoidal, which is analogous to an alternating voltage in electric circuit. An analogy was employed between the mass transfer for the facilitated transport with fixed site carrier membrane and the electron transfer in a single parallel resistor-capacitor (RC) circuit. The current model showed that the facilitation factor, F, increased linearly with the extent of the pressure fluctuation, α p (= p d p o ) , the ratio of the time scales of diffusion to chemical reaction, ψ(= k 2L 2 D ) , and the ratio of the total carrier concentration to the solute solubility in matrix, γ(= C B O C A ∞ ) . F also increased logarithmically with one plus the combined driving force for facilitation, Δ (= Kp 0), i.e. (1+ Δ). The model was examined against experimental data on oxygen transport in poly(dimethyl siloxane), poly(butyl methacrylate) and poly(methyl methacrylate) with metallo-porphyrin carrier and the agreement was exceptional.