Reconstitution of glucose-transporting vesicles from erythrocyte membranes disaggregated in detergent.

With the eventual aim of purifying a membrane transport system by using reconstitution of transport activity as an assay, I showed that if, after the erythrocyte membrane is solubilized in deoxycholate, the detergent is removed, membrane vesicles re-form which retain glucose-transport activity. They take up and release D-glucose in preference to L-glucose and the uptake and release are sensitive to Hg2+ and phloretin. Release of tracer D-glucose is competitively inhibited by transported sugars inside the vesicles and increased by unlabelling D-glucose in the outside medium. Uptake of tracer is increased so much by preloading vesicles with unlabelled transported sugars that the tracer is probably concentrated against a gradient. When the membrane is solubilized, two proteins that span the membrane can be separated, suggesting that it will be possible to fractionate the membrane before reconstitution.     

The inactivation by fluorodinitrobenzene of glucose transport across the human erythrocyte membrane The effect of glucose inside or outside the cell

The inactivation of glucose transport in human red cells by fluorodinitrobenzene is accelerated by 120 mM glucose outside the cell but retarded at least 50% by 120 mM glucose inside the cell. This suggests that the transport system is predominantly in one conformation when there is glucose inside the cell, and in another conformation when there is glucose outside the cell.     

Equilibrium ligand binding to the human erythrocyte sugar transporter. Evidence for two sugar-binding sites per carrier

Equilibrium [3H]cytochalasin B binding to class I sites of human red cell membranes (the sugar transporter) was examined in the presence and absence of intracellular or extracellular sugars known to interact with the transport system. D-Glucose, a transported sugar, is without effect on cytochalasin B binding when present in the extracellular medium but is an effective inhibitor of binding when present within the cell. Ethylidene glucose and maltose (reactive but nontransported sugars) inhibit cytochalasin B (CCB) binding when present either outside or inside the red cell. Inhibition by intracellular sugar (Si) is of the simple, linear competitive type. Inhibition by extracellular sugars (So) is more complex; the Kd(app) for cytochalasin B binding increases in a saturable fashion with [So]. These observations are compared with the predictions of the one-site, alternating conformer model and the two-site model for substrate binding to the sugar transporter, X. The experimental results are inconsistent with the one-site model but are explained by a two-site model in which the ternary complexes of So . X . Si or So . X . CCBi exist and where the binding sites for So and Si display negative cooperativity when occupied by nontransported substrate and little or no cooperativity when occupied by the transported species, D-glucose.     

Computer modelling approach to study the modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein

The modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein (ABP) have been studied by energy minimization using the low resolution (2.4 A) X-ray data of the protein. These studies suggest that these sugars preferentially bind in the alpha-form to ABP, unlike L-arabinose where both alpha- and beta-anomers bind almost equally. The best modes of binding of alpha- and beta-anomers of D-galactose and D-fucose differ slightly in the nature of the possible hydrogen bonds with the protein. The residues Arg 151 and Asn 232 of ABP from bidentate hydrogen bonds with both L-arabinose and D-galactose, but not with D-fucose or D-glucose. However in the case of L-arabinose, Arg 151 forms hydrogen bonds with the hydroxyl group at the C-4 atom and the ring oxygen, whereas in case of D-galactose it forms bonds with the hydroxyl groups at the C-4 and C-6 atoms of the pyranose ring. The calculated conformational energies also predict that D-galactose is a better inhibitor than D-fucose and D-glucose, in agreement with kinetic studies. The weak inhibitor D-glucose binds preferentially to one domain of ABP leading to the formation of a weaker complex. Thus these studies provide information about the most probable binding modes of these sugars and also provide a theoretical explanation for the observed differences in their binding affinities.     

Interactions of sodium pentobarbital with D-glucose and L-sorbose transport in human red cells.

Pentobarbital acts as a mixed inhibitor of net D-glucose exit, as monitored photometrically from human red cells. At 30 degrees C the Ki of pentobarbital for inhibition of Vmax of zero-trans net glucose exit is 2.16+/-0.14 mM; the affinity of the external site of the transporter for D-glucose is also reduced to 50% of control by 1. 66+/-0.06 mM pentobarbital. Pentobarbital reduces the temperature coefficient of D-glucose binding to the external site. Pentobarbital (4 mM) reduces the enthalpy of D-glucose interaction from 49.3+/-9.6 to 16.24+/-5.50 kJ/mol (P<0.05). Pentobarbital (8 mM) increases the activation energy of glucose exit from control 54.7+/-2.5 kJ/mol to 114+/-13 kJ/mol (P<0.01). Pentobarbital reduces the rate of L-sorbose exit from human red cells, in the temperature range 45 degrees C-30 degrees C (P<0.001). On cooling from 45 degrees C to 30 degrees C, in the presence of pentobarbital (4 mM), the Ki (sorbose, glucose) decreases from 30.6+/-7.8 mM to 14+/-1.9 mM; whereas in control cells, Ki (sorbose, glucose) increases from 6.8+/-1.3 mM at 45 degrees C to 23.4+/-4.5 mM at 30 degrees C (P<0.002). Thus, the glucose inhibition of sorbose exit is changed from an endothermic process (enthalpy change=+60.6+/-14.7 kJ/mol) to an exothermic process (enthalpy change=-43+/-6.2 7 kJ/mol) by pentobarbital (4 mM) (P<0.005). These findings indicate that pentobarbital acts by preventing glucose-induced conformational changes in glucose transporters by binding to 'non-catalytic' sites in the transporter.     

Specificity and kinetics of hexose transport in Trypanosoma brucei

Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.     

Studies on the synthesis of valienamine and 1-epi-valienamine starting from D-glucose or L-sorbose

Two synthetic routes to a carbocyclic precursor to valienamine are reported, starting from either D-glucose or L-sorbose and using ring-closing metathesis as a key step. A low-yielding synthesis of 1-epi-valienamine is reported. Results from an abortive third possible route to valienamine based on an early introduction of nitrogen are discussed.     

Apparent noncompetitive inhibition of choline transport in erythrocytes by inhibitors bound at the substrate site

Summary According to the conventional carrier model, an inhibitor bound at the substrate transfer site inhibits competitively when on the same side of the membrane as the substrate, but noncompetitively when on the opposite side. This prediction was tested with the nonpenetrating choline analog dimethyl-n-pentyl (2-hydroxyethyl) ammonium ion. In zerotrans entry and infinitetrans entry experiments, where the labeled substrate and the inhibitor occupy the same compartment, the inhibition was competitive, but in zerotrans exit it was noncompetitive, in accord with the model. Similar behavior was seen with dimethyl-n-decyl (2-hydroxyethyl) ammonium ion. With this property of the choline transport system established, it becomes possible to estimate the relative affinity inside and outside of inhibitors present on both sides of the membrane. The tertiary amine, dibutylaminoethanol, which enters the cell by simple diffusion, is such an inhibitor. Here the inhibition kinetics were the reverse of those for nonpenetrating inhibitors; zerotrans and infinitetrans exit was inhibited competitively, and zerotrans entry noncompetitively. It follows that dibutylaminoethanol binds predominantly to the inner carrier form.     

Effects of inhibitor,competitors and temperature on transport of carbohydrate in Crithidia luciliae

Effects of KCN (10^?4 M), simultaneous presence of varying concentrations of D-glucose and L-sorbose, and temperature on transport of carbohydrate in C. luciliae have been studied. The rate of carbohydrate entrance is inhibited, in all sugars used, ranging from 19% to 70% inhibition at 0.5 mM external concentrations. However, this inhibitor does not affect transport from external concentrations of the order of 0.02 M. At 20 mM external concentration, the rate of L-sorbose entrance is greatly inhibited by the simultaneous presence of D-glucose, and the transport mechanism shows enormously greater affinity for glucose than for other monosaccharides. However, at 0.5 mM external concentration, the rate of sorbose entrance is not inhibited at all by the simultaneous presence of D-glucose. In the temperature interval 15°–25°C, the Q₁₀ for rate of entrance when the external concentration is 0.5 mM is 2.8 times larger than the Q₁₀ when the external concentration is 20 mM. These data are interpreted as strongly suggesting two mechanisms for carbohydrate entrance: (a) facilitated diffusion, of importance only at high external concentrations; (b) an active transport mechanism, active at low external concentrations and dependent upon a supply of metabolic energy. These results are compared with those reported in the literature for other types of cells.     

Transport of 3-O-methyl D-glucose and beta-methyl D-glucoside by rabbit ileum.

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereo-specific affinity and (b) are common. The apparent affinity constants at the brush-border indicate that sugars are selected in the order, beta-methyl glucose greater than D-galactose greater than 3-O-methyl glucose, (the Km values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the Kt values for Na+ activation of sugar entry across the brush-border are: 27, 25, and 140 mequiv. for beta-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na+, water, sugar and membrane-binding groups are all factors which determine selective affinity. In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer (Na) or (sugar) is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)2. This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion. Theophylline reduces the serosal border permeability to beta-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increase the intracellular accumulation of these sugars. The permeability of the serosal border to beta-methyl glucose entry is lower than permeability of the serosal border to beta-methyl glucose exit, which suggested that beta-methyl glucose may be convected out of the cell across the lateral serosal border.     

Chemotaxis towards sugars by Bacillus subtilis.

Many sugars and derivatives were tested in the capillary assay for their attraction of Bacillus subtilis. The major attractants were 2-deoxy-D-glucose, D-fructose, gentiobiose, D-glucose, maltose, D-mannitol, D-mannose, N-acetylglucosamine, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, N-acetylmannosamine, alpha-methyl-D-mannoside, D-sorbitol, L-sorbose, sucrose, trehalose and D-xylose. Only glucose chemotaxis was completely constitutive. Competition experiments were carried out to determine the specificities of chemoreceptors. There were 25 instances of no influence of two sugars on each other's taxis, 92 instances of one sugar interfering non-reciprocally with chemotaxis towards another and 49 instances of two sugars reciprocally competing. However, in most of the last instances, other sugars were identified that interfered with chemotaxis towards one member of the pair but not the other. Thus, nearly all sugars and related compounds appear to be detected by their own chemoreceptors, but many secondary interactions exist.     

An explanation of the asymmetric binding of sugars to the human erythrocyte sugar-transport systems (Short Communication)

6-O-Alkyl-d-galactoses competitively inhibit the erythrocyte sugar-transport system when added to the outside of the cells, but not to the inside. n-Propyl beta-d-glucopyranoside competitively inhibits the system on the inside of the cells, but not on the outside. A model for sugar transport is proposed.     

Synthesis and bioactivity of C-2 and C-3 methyl ether derivatives of brassinolide

The following six novel methyl ether derivatives of brassinolide were prepared and their brassinosteroid activity was measured by means of the rice leaf lamina inclination bioassay: 2-O-methylbrassinolide, 3-O-methylbrassinolide, 2,22,23-tri-O-methylbrassinolide, 3,22,23-tri-O-methylbrassinolide, 2-O-methyl-25-methoxybrassinolide and 3-O-methyl-25-methoxybrassinolide. Brassinolide was used as a standard for comparison. All six compounds were also tested in the presence of 1000 ng of indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids. The 2-O-methyl- and 3-O-methylbrassinolide derivatives showed weak activity at high doses, which was enhanced by IAA, especially in the case of the 3-O-methyl derivative. Similarly, the 2,22,23-tri-O-methyl- and 3,22,23-tri-O-methyl derivatives displayed weak bioactivity on their own, but significantly stronger activity when applied with IAA. The 3-O-methyl and 3,22,23-tri-O-methyl analogues plus IAA were comparable in bioacivity to brassinolide alone, but were less active than brassinolide plus IAA. In each case, O-methylation at C-2 resulted in a greater loss of activity than O-methylation at C-3 under the same conditions. The relatively strong activity of 3,22,23-tri-O-methylbrassinolide in the presence of IAA is especially noteworthy as it indicates that free hydroxyl groups at C-3, C-22, and C-23 are not essential for bioactivity. Finally, 2-O-methyl- and 3-O-methyl-25-methoxybrassinolide were essentially inactive alone, and showed only a modest increase in bioactivity when coapplied with IAA.     

Preparation and some properties of rat skeletal-muscle polyribosomes

1. A series of d-galactose derivatives substituted at C-1 and C-6 were tested for active accumulation by everted segments of hamster and rat intestine. 2. d-Galactose and 6-deoxy-6-fluoro-d-galactose were accumulated far more rapidly than 6-deoxy- and 6-chloro-6-deoxy-d-galactose, and this is interpreted as due to hydrogen-bonding at C-6 during the transport process. 3. 6-Bromo-6-deoxy- and 6-deoxy-6-iodo-d-galactose were not actively transported, indicating that the allowed size of substituent at C-6 lies between that of chlorine and bromine atoms. 4. Similar results were obtained at C-1. Both methyl alpha-d-galactopyranoside and methyl beta-d-galactopyranoside were well transported, but methyl beta-d-thiogalactopyranoside and 1-deoxy-d-galactose were not transported; d-galactopyranosyl fluoride was transported, but only poorly. Again hydrogen-bonding is suggested. 5. It is proposed that d-glucose is the ideal structure for active transport and that binding occurs at C-1, C-2, C-3, C-4 and C-6. Loss of two or more of these bonds usually causes loss of active transport. 6. By plotting Lineweaver-Burk plots of the rates of transport of the galactose derivatives, the apparent V and K(m) values were obtained. With hamster intestine both these values were very reproducible. Contrary to expectation, V varied for different sugars. 7. The K(i) of some of the analogues modified at C-1 and C-6 was determined with methyl alpha-d-glucoside as substrate. 8. An attempt to alkylate the carrier by using methyl 3,4-anhydro-alpha-d-galactoside was unsuccessful. There was no evidence that this compound was bound to the carrier.     

Asymmetric transport of D-glucose anomers across the human erythrocyte membrane

The anomeric preference in the influx and efflux of D-glucose across the human erythrocyte membrane was studied. beta-D-Glucose was transported 1.5 times faster than alpha-D-glucose into the cells, when washed cells were incubated at 20 degrees C in medium containing either alpha- or beta-D-glucose (100 mM). On the other hand, no difference between half-times of efflux of the two anomers was distinguishable. The result demonstrates the presence of influx-efflux asymmetry in anomeric preference in D-glucose transport across the human erythrocyte membrane, and is consistent with the view (Barnett et al., Biochem. J. 145, 417-429, 1975) that the C-1 hydroxyl group of D-glucose interacts with the D-glucose transport protein only in the influx, but not in the efflux.     

Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes

Summary The sidedness of phloretin binding to the glucose carrier has been determined by comparing the type of inhibition produced in zerotrans entry and zerotrans exit experiments. Initial rates of zerotrans entry were measured by the method of R.D. Taverna and R.G. Langdon (Biochim. Biophys. Acta 298:412–421, 1973), which involves pink ghosts loaded with glucose oxidase; this obviates the problem of rapid substrate accumulation inside the cells. With phloretin equilibrated across the membrane, the inhibition of entry was competitive, and the inhibition of exit noncompetitive. The experimental procedures were validated by showing that the inhibition by cytochalasin B, known to bind inside but not outside, was noncompetitive in entry and competitive in exit, as predicted. It was also demonstrated that even after pre-incubation of the cells with a relatively high concentration of phloretin, the phloretin adsorbed in the membrane did not significantly alter the rate of carrier reorientation. The results show that the outward-facing form of the glucose carrier, but not the inward-facing form, bears a phloretin binding site; thus phloretin, as well as cytochalasin B, is bound asymmetrically, phloretin outside and cytochalasin B inside.     

Characteristics of D-galactose transport systems by luminal membrane vesicles from rabbit kidney

The characteristics of renal transport of D-galactose by luminal membrane vesicles from either whole cortex, pars recta or pars convoluta of rabbit proximal tubule were investigated by a spectrophotometric method using a potential-sensitive carbocyanine dye. Uptake of D-galactose by luminal membrane vesicles prepared from whole cortex was carried out by an Na+-dependent and electrogenic process. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for the uptake of D-galactose. Tubular localization of the transport systems was studied by the use of vesicles derived from pars recta and pars convoluta. In pars recta, Na+-dependent transport of D-galactose and D-glucose occurred by means of a high-affinity system (half-saturation: D-galactose, 0.15 +/- 0.02 mM; D-glucose, 0.13 +/- 0.02 mM). These results indicated that the "carrier' responsible for the uptake of these hexoses does not discriminate between the steric position of the C-4 hydroxyl group of these two isomers. This is further confirmed by competition experiments, which showed that D-galactose and D-glucose are taken up by the same and equal affinity transport system by these vesicle preparations. Uptake of D-galactose and D-glucose by luminal membrane vesicles isolated from pars convoluta was mediated by a low-affinity common transport system (half-saturation: D-galactose, 15 +/- 2 mM; D-glucose, 2.5 +/- 0.5 mM). These findings strongly suggested that the "carrier' involved in the transport of monosaccharides in vesicles from pars convoluta is specific for the steric position of the C-4 hydroxyl group of these sugars and presumably interacts only with D-glucose at normal physiological concentration.     

Evidence for non-uniform distribution of D-glucose within human red cells during net exit and counterflow

The kinetic parameters of net exit of D-glucose from human red blood cells have been measured after the cells were loaded to 18 mM, 75 mM and 120 mM at 2 degrees C and 75 mM and 120 mM at 20 degrees C. Reducing the temperature, or raising the loading concentration raises the apparent Km for net exit. Deoxygenation also reduces the Km for D-glucose exit from red blood cells loaded initially to 120 mM at 20 degrees C from 32.9 +/- 2.3 mM (13) with oxygenated blood to 20.5 +/- 1.3 mM (17) (P less than 0.01). Deoxygenation increases the ratio Vmax/Km from 5.29 +/- 0.26 min-1 (13) for oxygenated blood to 7.13 +/- 0.29 min-1 (17) for deoxygenated blood (P less than 0.001). The counterflow of D-glucose from solutions containing 1 mM 14C-labelled D-glucose was measured at 2 degrees C and 20 degrees C. Reduction in temperature, reduced the maximal level to which labelled D-glucose was accumulated and altered the course of equilibration of the specific activity of intracellular D-glucose from a single exponential to a more complex form. Raising the internal concentration from 18 mM to 90 mM at 2 degrees C also alters the course of equilibration of labelled D-glucose within the cell to a complex form. The apparent asymmetry of the transport system may be estimated from the intracellular concentrations of labelled and unlabelled sugar at the turning point of the counterflow transient. The estimates of asymmetry obtained from this approach indicate that there is no significant asymmetry at 20 degrees C and at 2 degrees C asymmetry is between 3 and 6. This is at least 20-fold less than predicted from the kinetic parameter asymmetries for net exit and entry. None of the above results fit a kinetic scheme in which the asymmetry of the transport system is controlled by intrinsic differences in the kinetic parameters at the inner and outer membrane surface. These results are consistent with a model for sugar transport in which movement between sugar within bound and free intracellular compartments can become the rate-limiting step in controlling net movement into, or out of the cell.     

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]

In mechanistic studies by isotope-exchange tecniques of the conversion of D-fructose and D-glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D-glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D-glucose-2-³H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D-glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect (k_h/k_t=4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D-glucose to D-fructose, as evidenced by the conversion of D-glucose-2-³H into D-fructose-1-³H in acidified water.