Artificial enzymic membrane pump for glucose transport against its chemical gradient

An artificial glucokinase/phosphatase porous membrane separating two unequal compartments of a diffusion cell was used to pump glucose from one compartment against the concentration gradient of the opposite one. Our results mainly demonstrate that, a kinase/phosphatase reactional sequence acting on both parts of a pore structure and necessarily in unstirred layers may pump a neutral solute from one compartment against the concentration gradient of the opposite one without any detectable pollution of the charged intermediary product. The corresponding theoretical analysis, which underlines the key role played by the diffusion layers located on both parts of this bienzymic membrane and the membrane's charge effect, was found in good agreement with the experimental data. This study corroborates well the new kinetic model recently proposed for primary scalar active transport of small hydrophilic molecules involving ATP as the energy supply.     

ATP-dependent active transport simulations based on a phosphatase-channel-kinase membrane structure

Simulations of coupled interactions involving enzymatic reaction diffusion and electrostatic interactions were conducted under a fixed phosphatase-channel-kinase (PCK) topology oriented from the outside to the inside of a charged membrane structure. Depending on the phosphatase and kinase locations, we recently demonstrated that active transport of a phosphorylated substrate may occur via this PCK topology. The present analysis demonstrates that, if in addition to this topology, a phosphatase activity (P(1)) is also present on the inner side of the membrane, but outside the unstirred layer surrounding the inner membrane surface, then active transport of the corresponding unphosphorylated substrate may also occur. Therefore, this PCK membrane topology, which behaves as a specific ATP-dependent transporter, appears as a general topology permitting; first, on its own the active transport of a phosphorylated substrate; second, when associated with a phosphatase acting in the bulk of the receiver compartment, the active transport of the corresponding unphosphorylated substrate, that is, in most cases, the transport of an uncharged substrate. The general mathematical model given permits the active transport of a phosphorylated substrate to be analyzed (in the absence of P(1)), the active transport of an unphosphorylated substrate (in the presence of P(1)), whatever the charge distributions on both sides of the membrane surface and whatever the positions of the membrane-bound phosphatase and the membrane-bound kinase. This general model also takes into account the consumption of ATP occurring into the receiver compartment during the time course of these transport phenomena. A broad analysis of the role played by the main parameters taken into account in the model was conducted to precisely define the physicochemical conditions and the membrane topology needed for the highest active transports within the shortest time.     

Simulations of the active transport of a neutral solute based on a kinase-channel-phosphatase topology

Simulations of coupled interactions involving two opposite enzymatic reactions, solute diffusions, and electrostatic interactions between membrane charges and charged solutes were conducted under a fixed kinase-channel-phosphatase (KCP) topology oriented from the outside to the inside of a porous membrane structure. Depending on the kinase and phosphatase locations, we recently demonstrated that an active transport of a phosphorylated substrate may occur via the opposite topology, that is, a PCK topology. The present analysis demonstrates that, under a KCP membrane topology, which also behaves as a specific ATP-dependent transporter, the active transport of a neutral substrate may occur. This analogous active transport appears to be dependent on the phosphatase location and on the membrane surface potentials. A broad analysis of the role played by the main parameters taken into account in the model was conducted in order to define precisely the physico-chemical conditions and the membrane topology needed for the highest active transports within the shortest time.     

The molecular mechanism of dicarboxylic acid transport in Escherichia coli K 12.

It is the purpose of this communication to review the properties of the dicarboxylic acid transport system in Escherichia coli K 12, in particular the role of various dicarboxylate transport proteins, and the disposition of these components in the cytoplasmic membrane. The dicarboxylate transport system is an active process and is responsible for the uptake of succinate, fumarate, and malate. Membrane vesicles prepared from the EDTA, lysozyme, and osmotic shock treatment take up the dicarboxylic acids in the presence of an electron donor. Genetic analysis of various transport mutants indicates that there is only one dicarboxylic acid transport system present in Escherichia coli K 12, and that at least 3 genes, designated cbt, dct A, and dct B, are involved in this transport system. The products corresponding to the 3 genes are: a periplasmic binding protein (PBP) specified by cbt, and 2 membrane integral proteins, SBP 1 and SBP 2, specified by dct B and dct A, respectively. Components SBP 1 and SBP 2 appear to be exposed on both the inner and outer surfaces of the membrane, and lie in close proximity to each other. The substrate recognition sites of SBP 2 and SBP 1 are exposed on the outer and inner surfaces of the membrane respectively. The data presently available suggest that dicarboxylic acids may be translocated across the membrane via a transport channel. A tentative working model on the mechanism of translocation of dicarboxylic acids across the cell envelope by the periplasmic binding protein, and the 2 membrane carrier proteins is presented.     

Solute molecular transport through polyimide asymmetric organic solvent nanofiltration (OSN) membranes and the effect of membrane-formation parameters on mass transfer

This work studies the effect of two membrane-formation parameters, evaporation time and casting thickness, on the diffusive mass transport of organic solutes through an organic solvent nanofiltration (OSN) membrane. These parameters showed a coupled effect on the final membrane thickness, which was explained in terms of top-layer formation. In a concentration-driven dialysis, both parameters, as well as the resulting membrane thickness, had a significant effect on mass transport. Casting thickness had the greatest effect on membrane mass transport rates. Multivariate regression was used to model the dialysis process with acceptable fit. A representation of the membrane morphology was obtained from SEM pictures and used to formulate an alternative mechanistic mass-transport model. A resistance-circuit analogy was used to describe transport through the top and microporous layers, which also considered diffusion through the pores and the polymer for each layer. From the analyses of the models and considering that no differences in top-layer thickness were observed by SEM, it is concluded that membrane asymmetry, determined by the formation parameters, controls mass transport, rather than top-layer thickness.     

Some aspects of the active transport of amino acids

Active transport is the transport of substances against their activity gradients. In the cell, metabolic processes provide the energy for active transport through the membrane, during which amino acids are coupled with a carrier system located in the membrane. Some of the proteins involved in the translocation of amino acids have now been isolated from E. coli.     

Electrochemical characterization of an asymmetric nanofiltration membrane with NaCl and KCl solutions: influence of membrane asymmetry on transport parameters

Electrochemical characterization of a nanofiltration asymmetric membrane was carried out by measuring membrane potential, salt diffusion, and electrical parameters (membrane electrical resistance and capacitance) with the membrane in contact with NaCl and KCl solutions at different concentrations (10(-3)< or =c(M)< or =5 x 10(-2)). From these experiments characteristic parameters such as the effective concentration of charge in the membrane, ionic transport numbers, and salt and ionic permeabilities across the membrane were determined. Membrane electrical resistance and capacitance were obtained from impedance spectroscopy (IS) measurements by using equivalent circuits as models. This technique allows the determination of the electrical contribution associated with each sublayer; then, assuming that the dense sublayer behaves as a plane capacitor, its thickness can be estimated from the capacitance value. The influence of membrane asymmetry on transport parameters have been studied by carrying out measurements for the two opposite external conditions. Results show that membrane asymmetry strongly affects membrane potential, which is attributed to the Donnan exclusion when the solutions in contact with the dense layer have concentrations lower than the membrane fixed charge (X(ef) approximately -0.004 M), but for the reversal experimental condition (high concentration in contact with the membrane dense sublayer) the membrane potential is practically similar to the solution diffusion potential. The comparison of results obtained for both electrolytes agrees with the higher conductivity of KCl solutions. On the other hand, the influence of diffusion layers at the membrane/solution interfaces in salt permeation was also studied by measuring salt diffusion at a given NaCl concentration gradient but at five different solutions stirring rates.     

Coupled transport membranes II: : The mechanism of uranium transport with a tertiary amine

This paper describes the parameters controlling the coupled transport of uranium anions through liquid membranes. The membranes consist of a microporous polymeric support with a liquid, tertiary amine complexing agent held within the pores by capillary forces. When this liquid membrane is interposed between two aqueous solutions of unequal ion concentrations, the complexing agent can pick up the anion on one side of the membrane and carry it across the membrane by diffusion in the form of a neutral complex. Ions of opposite charge may be carried in the same direction, or ions of like charge may be carried in the opposite direction. We refer to these two modes of transport as “co-transport” and “counter-transport”, respectively. In the coupled transport of uranium, both co-transport and counter-transport can occur. p]The coupling of the flows of two ions permits one of the ions to be pumped against its concentration gradient. We have demonstrated “uphill diffusion” of uranium against substantial concentration gradients, and at significant rates. A number of factors affect uranium flux, principally the concentrations of uranium and the coupled ion in the aqueous solutions. The base strength of the tertiary amine is also an important parameter.     

Enzymes immobilized on montmorillonite: comparison of performance in batch and packed-bed reactors

Summary The enzymes a-amylase, invertase and glucoamylase were immobilized on acid activated montmorillonite using two techniques, viz. adsorption and covalent binding, and their activities were tested in a batch and packed-bed reactor and were compared. The packed-bed reactor showed an improved performance for all immobilized enzymes, which was attributed to lowering of diffusional restrictions to mass transfer. Lower activity in case of batch reactor for immobilized invertase was due to a combined effect of loss of native conformation of enzyme on account of immobilization and mass transfer resistances due to improper diffusion of substrate to the active site of enzyme. For immobilized glucoamylase, the packed-bed reactor demonstrated exceptionally high activity that was very close to the free enzyme. Covalently bound glucoamylase showed higher activity than the free enzyme.     

Modeling of diffusive transport of benzoic acid through a liquid membrane

A model of diffusive transport of benzoic acid through a liquid membrane (LM) separating two aqueous solutions, based on diffusion layers and the assumption of a steady state, has been developed and tested using experimental results. It has been found that a model with the apparent partition coefficient dependent on the concentration is able to describe the time dependence of acid concentration in LM with and without a maximum on that dependence. The quality of the model fit with the single apparent diffusion coefficient of benzoic acid is the same as the one which takes into account the diffusion of benzoic acid in different forms (undissociated and dissociated form in aqueous phase, monomer and dimer in organic phase); however, in the second case, the model becomes overparameterized. Assuming that the partition and diffusion coefficients are constant, the diffusion layer model corresponds to the model of reversible consecutive reactions. Analytical solution for such case is given. Apart from the partition equilibrium, also kinetics of partitioning was considered. It was shown that in some basic situations both cases yield identical results.     

Stationary electrodiffusion–adsorption processes in membranes including diffuse double layer effects: A network approach

Ion transport across membranes with surface charge due to ion adsorption, including the diffuse double layer effects, is analysed using the network simulation method. The membrane system under study is a multilayer one constituted by a membrane and two diffusion boundary layers on both sides of the membrane. The ion transport processes are described by the Nernst–Planck and Poisson equations not only in the membrane–solution interfaces, but also in the membrane bulk and in the two diffusion boundary layers. The membrane has a negative surface charge due to an anion adsorption process. The structure of the equilibrium diffuse double layers and the steady-state current–voltage characteristic have been analysed for the case of an adsorption process described by a Langmuir-type adsorption isotherm. The evolution of the electric potential difference across the membrane system in the equilibrium state of the system as a function of the bathing concentrations, have been also analysed.     

Simulation of concurrent transport of counterions through a heterogeneous ion-exchange membrane

Numerical solutions of the problem of concurrent transport of two counterions in an ion-exchange membrane are presented and discussed with allowance made for the membranes heterogeneous structure. Densities of fluxes of co-ions and competing counterions are computed as a function of the current density and boundary concentrations on either side of the membrane. The obtain result is viewed as the initial product for solving multilayered problems containing membranes of other nature and diffusion layers surrounding the membrane.Translated from Elektrokhimiya, Vol. 41, No. 3, 2005, pp. 356–366.Original Russian Text Copyright © 2005 by Gnusin.     

Solvent-extraction and Langmuir-adsorption-based transport in chemically functionalized nanopore membranes

We have investigated the transport properties of nanopore alumina membranes that were rendered hydrophobic by functionalization with octadecyltrimethoxysilane (ODS). The pores in these ODS-modified membranes are so hydrophobic that they are not wetted by water. Nevertheless, nonionic molecules can be transported from an aqueous feed solution on one side of the membrane, through the dry nanopores, and into an aqueous receiver solution on the other side. The transport mechanism involves Langmuir-type adsorption of the permeating molecule onto the ODS layers lining the pore walls, followed by solid-state diffusion along these ODS layers; we have measured the diffusion coefficients associated with this transport process. We have also investigated the transport properties of membranes prepared by filling the ODS-modified pores with the water-immiscible (hydrophobic) liquid mineral oil. In this case the transport mechanism involves solvent extraction of the permeating molecule into the mineral oil subphase confined with the pores, followed by solution-based diffusion through this liquid subphase. Because of this different transport mechanism, the supported-liquid membranes show substantially better transport selectivity than the ODS-modified membranes that contain no liquid subphase.     

Boundary layer effect on product flux-splitting in an immobilized enzyme membrane system

An asymmetrical system involving glucose oxidase immobilized on the surface of a porous collagen membrane was studied. The enzyme was bound on only one side of a membrane used to separate two chambers of a diffusion cell. The volumes of these chambers were very different, and the medium in each chamber could be stirred independently. With the enzyme adjacent to the larger chamber, monitoring the building up of product in the two chambers revealed that the concentration was always higher in the receiving chamber than in the donating chamber, where the reaction occurred. This phenomenon can be explained by the high concentration of product in the immobilized enzyme microenvironment and by the role played by the diffusion layer existing close to the membrane in the reaction chamber affecting product flux-splitting. The thickness of this layer and the product concentration at the enzyme level were calculated for several hydrodynamic conditions in the enzymatic chamber. The ratio of product concentration in both chambers may become larger than 10 and was found to be dependent on the thickness of the diffusion layer. These findings should be useful for the design of reaction-separation bioreactors and could tentatively be used to explain some interface phenomena in compartmented reactions occurring in vivo.     

Diketopiperazines as a tool for the study of transport across the blood-brain barrier (BBB) and their potential use as BBB-shuttles

Here we prepared and evaluated two libraries of mono-N-methylated and di-N-methylated diketopiperazines (DKPs) by parallel artificial membrane permeability assay and immobilized artificial membrane chromatography in order to obtain information on the features that govern the passage of peptidic molecules across the blood-brain barrier (BBB) by passive diffusion. On the basis of the results from these two libraries, we prepared and evaluated several DKP-baicalin and DKP-dopamine constructs. The DKPs or cyclic dipeptide scaffolds can be considered a novel family of brain delivery systems (BBB-shuttles) to transport to the brain drugs and other cargos that cannot cross the BBB unaided.     

Enhancement of membrane transport by spatial nonuniformity

Spatial nonuniformity in membranes can lead to enhanced flux. We show that this phenomenon exists in systems which obey nonlinear transport laws. Examples include carrier-mediated transport, electrically facilitated transport and Fickian diffusion in systems with concentration-dependent diffusion coefficients. The latter exists if binding of the transported species to the membrane matrix occurs. This may contribute to the enhanced current efficiency in perfluorinated membranes of the Nafion type.     

Synthesis and transport abilities of new membrane materials incorporating mono- and bi-pyrazolic compounds

The membrane materials were obtained by photopolymerisation of formulation that contain the active monomer spread on a polyacrylonitrile support. The facilitated transport and the extraction power of Cd(II), Pb(II) and Hg(II) through the synthesised membranes were reported. We have determined both the diffusion flux of different cations and the selectivity of the prepared membranes towards each cations.     

Layer-by-layer-assembled microfiltration membranes for biomolecule immobilization and enzymatic catalysis

Multilayer assemblies of polyelectrolytes, for protein immobilization, have been created within the membrane pore domain. This approach was taken for two reasons: (1) the high internal membrane area can potentially increase the amount of immobilized protein, and (2) the use of convective flow allows uniform assembly of layers and eliminates diffusional limitations after immobilization. To build a stable assembly, the first polyelectrolyte layer was covalently attached to the membrane surface and inside the pore walls. Either poly(L-glutamic acid) (PLGA) or poly(L-lysine) (PLL) was used in this step. Subsequent deposition occurs by multiple electrostatic interactions between the adsorbing polyelectrolyte [poly(allylamine) hydrochloride (PAH) or poly(styrenesulfonate) (PSS)] and the oppositely charged layer. Three-layer membranes were created: PLL-PSS-PAH or PLGA-PAH-PSS, for an overall positive or negative charge, respectively. The overall charge on both the protein and membrane plays a substantial role in immobilization. When the protein and the membrane are oppositely charged, the amount immobilized and the stability within the polyelectrolyte assembly are significantly higher than for the case when both have similar charges. After protein incorporation in the multilayer assembly, the active site accessibility was comparable to that obtained in the homogeneous phase. This was tested by affinity interaction (avidin-biotin) and by carrying out two reactions (catalyzed by glucose oxidase and alkaline phosphatase). Besides simplicity and versatility, the ease of enzyme regeneration constitutes an additional benefit of this approach.     

Analysis of the pressure-induced potential arising through composite membranes with selective surface layers

When a pressure gradient is applied through a charged selective membrane, the transmembrane electrical potential difference, called the filtration potential, results from both the applied pressure and induced concentration difference across the membrane. In this work we investigate the electrokinetic properties relative to both active and support layers of a composite ceramic membrane close to the nanofiltration range. First, the volume charge density of the active layer is obtained by fitting a transport model to experimental rejection rates (which are controlled by the active layer only). Next, the value of the volume charge density is used to compute the theoretical filtration potential through the active layer. For sufficiently high permeate volume fluxes, the concentration difference across the active layer becomes constant, which allows assessing the membrane potential of the active layer. Experimental measurements of the overall filtration potential arising through the whole membrane are performed. The contribution of the support layer to this overall filtration potential is put in evidence. That implies that the membrane potential of the active layer cannot be deduced directly from the overall filtration potential measurements. Finally, the contribution of the support layer is singled out by subtracting the theoretical filtration potential of the active layer from the experimental filtration potential measured across the whole membrane (i.e., support + active layers). The amphoteric behavior of both layers is put in evidence, which is confirmed by electrophoretic measurements carried out with the powdered support layer and by recently reported tangential streaming potential measurements.     

Liquid membrane transport: a survey

The literature pertaining to facilitated transport and liquid membrane separations is reviewed and summarized, especially work reported since 1977. Liquid membranes of all geometries are discussed, including immobilized liquid membranes and liquid surfactant or emulsion liquid membranes. Emphasis is placed on facilitated, or carrier-mediated transport in both configurations although other mechanisms such as coupled-transport and transport due to solubility differences are discussed. Mathematical modeling and analytical solutions for facilitated transport models are summarized. The possibility of industrial application of liquid membrane technology is mentioned and the most important experimental techniques for liquid membrane research are discussed. Also, directions for future research are recommended.