Transport of the highly charged myo-inositol hexakisphosphate molecule across the red blood cell membrane: a phase transfer and biological study

To address the problem of delivering highly charged small molecules, such as phytic acid (InsP(6) or IHP), across biological membranes, we investigated an approach based on a non-covalent interaction between transport molecule(s) and IHP. Thus, we synthesized a collection of compounds containing IHP ionically bound to lipophilic (but non-lipidic) ammonium or poly-ammonium cations. First, we assessed the ability of these water-soluble salts to cross a biological membrane by measuring the partition coefficients between human serum and 1-octanol. In view of the ability of IHP to act as potent effector for oxygen release, the O(2)-hemoglobin dissociation curves were then measured for the most efficient salts on whole blood. From both the biological and the physical properties of IHP-ammonium salts we determined that cycloalkylamines (or poly-amines) were the best transport molecules, especially cycloheptyl- and cyclooctylamine. Indeed, the octanol/serum partition coefficient of IHP undecacyclooctylammonium salt, is superior to 1, which is very favorable for potential uptake into the red blood cell membrane. A qualitative correlation was found between the partitioning experiments and the biological evaluations performed on whole blood.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO3, NaCl, Na2SO4, KF, KNO3, KCl, K2SO4, MgCl2, CaCl2, CrCl3, MnCl2, FeCl3, CoCl2, and CuCl2 were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 degrees C) in these cases. The slope of plots of specific conductance, kappa, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The kappa values of electrolytes follow the sequence for the anions: SO4(2-)>Cl->NO3->F- while that for the cations: K+>Na+>Ca2+>Mn2+>Co2+>Cu2+>Mg2+>Cr3+>Fe3+. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, kappa=(RT/Nh)exp[-DeltaH*/RT]exp[DeltaS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[kappaNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of DeltaS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high DeltaS* value associated with the high value of energy of activation, Ea, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of DeltaS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO₃, NaCl, Na₂SO₄, KF, KNO₃, KCl, K₂SO₄, MgCl₂, CaCl₂, CrCl₃, MnCl₂, FeCl₃, CoCl₂, and CuCl₂ were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 °C) in these cases. The slope of plots of specific conductance, κ, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The κ values of electrolytes follow the sequence for the anions: SO₄²⁻>Cl⁻>NO₃⁻>F⁻ while that for the cations: K⁺>Na⁺>Ca²⁺>Mn²⁺>Co²⁺>Cu²⁺>Mg²⁺>Cr³⁺>Fe³⁺. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, κ=(RT/Nh)exp[−ΔH*/RT]exp[ΔS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[κNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of ΔS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high ΔS* value associated with the high value of energy of activation, E_a, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of ΔS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Transport across the bacterial outer membrane

Diffusion of small molecules across the outer membrane of gram-negative bacteria may occur through protein channels and through lipid bilayer domains. Among protein channels, many examples of trimeric porins, which produce water-filled diffusion channels, are known. Although the channels are nonspecific, the diffusion rates of solutes are often drastically affected by their gross physicochemical properties, such as size, charge, or lipophilicity, because the channel has a dimension not too different from that of the diffusing solutes. In the last few years, the structures of three such porins have been solved by X-ray crystallography. It is now known that a monomer unit traverses the membrane 16 times as -strands, and one of the external loop folds back into the channel to produce a narrow constriction. Most of the static properties of the channel, such as the pore size and the position of the amino acids that produce the constriction, can now be explained by the three-dimensional structure. Controversy, however, still surrounds the issue of whether there are dynamic modulation of the channel properties in response to pH, ionic strength, or membrane potential, and of whether such responses are physiological. More recently, two examples of monomeric porins have been identified. These porins allow a very slow diffusion of solutes, but the reason for this low permeability is still unclear. Finally, channels with specific binding sites facilitate the diffusion of specific classes of nutrients, often those compounds that are too large to penetrate rapidly through the porin channels. Lipid bilayers in the outer membrane were shown to be perhaps 50- to 100-fold less permeable to uncharged, lipophilic molecules in comparison with the bilayers made of the usual glycerophospholipids. This is caused by the presence of a lipopolysaccharide leaflet in the bilayer, and more specifically, by the presence of a larger number of fatty acids in each lipid molecule, and by the absence of unsaturated fatty acids in the lipopolysaccharide structure.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.     

Proton transport across charged membrane and pH oscillations.

Based on Eyring's multibarrier activation process, a mathematical model and equation is developed to account for proton diffusion through an immobilized protein and enzyme membrane perfused with an electrolyte, substrate, and a buffer. With this model we find that, in the presence of a buffer, our solution approaches the continuum case very rapidly. We apply our model to membranes composed of papain and bovine serum albumin and find that our theory closely stimulates the experimental observations on the effect of salt and buffer on proton diffusion. Our theory shows that the pH oscillations observed in the diffusion controlled papain-benzoyl-L-arginine ethyl ester (BAEE) reaction may be the result of CO2 dissolved in the bath at high pH. In our theory, under certain conditions and in agreement with experimental observation, the buffer penetration depth oscillates near the boundary of a papain membrane in a solution containing BAEE and borate. We also find that at low ionic strength small ions as well as a buffer are seen to oscillate if a membrane is highly charged.     

Transport of 3-bromopyruvate across the human erythrocyte membrane

3-Bromopyruvic acid (3-BP) is a promising anticancer compound because it is a strong inhibitor of glycolytic enzymes, especially glyceraldehyde 3-phosphate dehydrogenase. The Warburg effect means that malignant cells are much more dependent on glycolysis than normal cells. Potential complications of anticancer therapy with 3-BP are side effects due to its interaction with normal cells, especially erythrocytes. Transport into cells is critical for 3-BP to have intracellular effects. The aim of our study was the kinetic characterization of 3-BP transport into human erythrocytes. 3-BP uptake by erythrocytes was linear within the first 3 min and pH-dependent. The transport rate decreased with increasing pH in the range of 6.0–8.0. The K_m and V_m values for 3-BP transport were 0.89 mM and 0.94 mmol/(l cells x min), respectively. The transport was inhibited competitively by pyruvate and significantly inhibited by DIDS, SITS, and 1-cyano-4-hydroxycinnamic acid. Flavonoids also inhibited 3-BP transport: the most potent inhibition was found for luteolin and quercetin.     

Transport of macromolecules across arterial wall in the presence of local endothial injury.

The theoretical study of the subendothelial spread of macromolecules in the vicinity of a localized endothelial damage is of importance because of the large increased uptake of macromolecules which has been observed experimentally in certain regions of the arterial tree as compared to other regions. It has been hypothesized that the locally observed increased permeability may be due to endothelial injury produced by naturally occurring hemodynamic factors. The two-phase arterial wall model developed by Weinbaum &; Caro (1976) has been extended to obtain analytic solutions for the time dependent and steady state concentration distributions, flux and uptake in the arterial wall as a function of damage size, fraction of damaged surface and position from the damage. It has been demonstrated that with as little as 3% of the endothelial., surface damaged with locally spread holes of size 0·1 the thickness of the arterial media, the total uptake can increase by a factor of 2·5 over that of an artery with no endothelial damage.     

Transport of primary metabolites across the plant vacuolar membrane

Mesophyll cells and most types of storage cells harbor large central vacuoles representing the main cellular store for sugars and other primary metabolites like carboxylic- or and amino acids. The general biochemical characteristics of sugar transport across the vacuolar membrane are already known since a couple of years but only recently the first tonoplast sugar carriers have been identified on the molecular level. A candidate sucrose carrier has been identified in a proteomic approach. In Arabidopsis, the tonoplast monosaccharide transporters (TMT) represent a small protein family comprising only three members, which reside in the vacuolar membrane. Two of three tmt genes are induced upon cold, drought or salt stress and tmt knock out mutants exhibit altered monosaccharide levels upon cold induction. These observations indicate that TMT proteins represent the first examples of tonoplast sugar carriers involved in the cellular response upon osmotic stress stimuli.     

Microcalorimetry of biological macromolecules

The capabilities of contemporary differential scanning and isothermal titration microcalorimetry for studying the thermodynamics of protein unfolding/refolding and their association with partners, particularly target DNA duplexes, are considered. It is shown that the predenaturational changes of proteins must not be ignored in studying the thermodynamics of formation of their native structure and their complexes with partners, particularly their cognate DNA duplexes.     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Transport of fatty acids and metabolites across the peroxisomal membrane

The peroxisomal membrane forms a permeability barrier for a wide variety of metabolites required for and formed during fatty acid beta-oxidation. To communicate with the cytoplasm and mitochondria, peroxisomes need dedicated proteins to transport such hydrophilic molecules across their membranes. Genetic and biochemical studies in the yeast Saccharomyces cerevisiae have identified enzymes for redox shuttles as well as the first peroxisomal membrane transporter. This peroxisomal ATP-binding cassette transporter (Pat) is highly homologous to the gene mutated in X-linked adrenoleukodystrophy (X-ALD). The yeast Pat is required for import of activated fatty acids into peroxisomes suggesting that this is the primary defect in X-ALD.     

Transport of phage P22 DNA across the cytoplasmic membrane

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into Salmonella is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of ³²P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of Salmonella.     

Diffusion and diffusion-osmosis models of the transfer of charged molecules across biological barriers

Mathematical models of the transfer of large enough charged molecules (macroions) have been constructed on the basis of the classical equations of electromigration diffusion (Helmholtz-Smoluchowski, Goldman, and Goldman-Hodgkin-Katz). It is shown that ion transfer in placental barriers (mimicking lipid-protein membrane barriers) and in muscle barriers proceeds by different mechanisms. In placental barriers, the electromigration diffusion takes place through lipid-protein channels formed by conformational alteration of phospholipid and protein molecules, with diffusion coefficients D = (2.6–3.6) × 10⁻⁸ cm²/s. The transfer in muscle barriers is due to migration via charged interfibrillar channels with negative diffusion activation energy (explained by changes in the structure of muscle fibers and expenditures of thermal energy for the displacement of Cl⁻ from channel walls), and D = (6.0–10.0) × 10⁻⁶ cm²/s.     

Electrostatic interactions across a charged lipid bilayer

We present theoretical work in which the degree of electrostatic coupling across a charged lipid bilayer in aqueous solution is analyzed on the basis of nonlinear Poisson–Boltzmann theory. In particular, we consider the electrostatic interaction of a single, large macroion with the two apposed leaflets of an oppositely charged lipid bilayer where the macroion is allowed to optimize its distance to the membrane. Three regimes are identified: a weak and a high macroion charge regime, separated by a regime of close macroion–membrane contact for intermediate charge densities. The corresponding free energies are used to estimate the degree of electrostatic coupling in a lamellar cationic lipid–DNA complex. That is, we calculate to what extent the one-dimensional DNA arrays in a sandwich-like lipoplex interact across the cationic membranes. We find that, in spite of the low dielectric constant inside a lipid membranes, there can be a significant electrostatic contribution to the experimentally observed cross-bilayer orientational ordering of the DNA arrays. Our approximate analytical model is complemented and supported by numerical calculations of the electrostatic potentials and free energies of the lamellar lipoplex geometry. To this end, we solve the nonlinear Poisson–Boltzmann equation within a unit cell of the lamellar lipoplex using a new lattice Boltzmann method. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.