Effects of membrane steroid modification on human erythrocyte glucose transport

The partial removal of cholesterol from the human erythrocyte membrane, by contact with lecithin sols, had mixed effects on the transport of d-glucose. When about 8% of the cholesterol was removed, the rate of d-glucose transfer was increased, but as cholesterol was progressively further removed, the transport was inhibited. Replacement of the depleted cholesterol by 3-ketosteroids did not restore the transport activity; but with substitution of steroids containing only a 3β-hydroxy substituent, the rate of glucose transport returned to normal. In some instances, as little as a 2% replacement of the removed cholesterol by 3β-hydroxy steroids was sufficient for full restoration of d-glucose transport. Cholesterol substitution by steroids with a more planar nucleus and a more bulky side chain than cholesterol also aided in the restoration of glucose transfer. The partial removal of cholesterol had no effect on the apparent K_m for d-glucose, but excessive membrane cholesterol led to a 4-fold decrease in d-glucose affinity. The extent of transport inhibition by a fixed phloretin treatment was independent of membrane steroid content.     

Asymmetric transport of a fluorescent glucose analogue by human erythrocytes

A fluorescent glucose analogue, 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (NBDG), was synthesized and its interactions with the hexose transport system of the human red blood cell were investigated. NBDG entry is inhibited by increasing concentrations of d-glucose (K_i = 2 mM). However, NBDG exit is unaffected by d-glucose in red blood cells. Cytochalasin B was found to inhibit both NBDG entry and exit. NBDG accumulates in the red blood cell above the theoretical equilibrium concentration. Accumulation of NBDG is temperature-sensitive and is due to the binding of NBDG to some intracellular substance. The binding of NBDG to purified hemoglobin suggests that accumulation of NBDG by erythrocytes is due to the intracellular binding of NBDG to hemoglobin. NBDG does not accumulate in pink erythrocyte ghosts, while its rate of uptake is still inhibited by d-glucose and cytochalasin B. Although there was no apparent d-glucose inhibition of NBDG exit by intact red blood cells, d-glucose was able to inhibit NBDG exit by pink erythrocyte ghosts. The differing properties of NBDG influx and efflux support the interpretation that the hexose transport system of the human red blood cell appears asymmetric although it may be intrinsically symmetric.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Substrate specificity of a chimera made from Xenopus SGLT1-like protein and rabbit SGLT1

To characterize the sugar translocation pathway of Na⁺/glucose cotransporter type 1 (SGLT1), a chimera was made by substituting the extracellular loop between transmembrane domain (TM) 12 and TM13 of Xenopus SGLT1-like protein (xSGLT1L) with the homologous region of rabbit SGLT1. The chimera was expressed in Xenopus oocytes and its transport activity was measured by the two-microelectrode voltage-clamp method. The substrate specificity of the chimera was different from those of xSGLT1L and SGLT1. In addition the chimera's apparent Michaelis-Menten constant (K_m) for myo-inositol, 0.06 mM, was about one fourth of that of xSGLT1L, 0.25 mM, while the chimera's apparent K_m for d-glucose, 0.8 mM, was about one eighth of that of xSGLT1L, 6.3 mM. Our results suggest that the extracellular loop between TM12 and TM13 participates in the sugar transport of SGLT1.     

The utilization of bathocuproinedisulfonic acid as a reagent for determining D-glucose and D-galactose levels in glycoconjugates

A sensitive, rapid, and reliable method for measuring d-glucose and d-galactose levels in glycoconjugates has been developed. In this method, the NAD(P)H produced from the enzymatic oxidation of the monosaccharides is reacted with a CuSO₄-bathocuproinedisulfonic acid reagent (Cu-BCS) to produce a color complex absorbing maximally at 486 nm. With galactose dehydrogenase and glucose dehydrogenase serving as the model enzymes, graphs of absorbance versus varying d-glucose or d-galactose concentrations yielded a linear plot from 2.5 to 250 nmol of sugar. Using this procedure, sugar released by acid hydrolysis from lactose, porcine submaxillary mucin and raffinose was quantified. When p-nitrophenyl-α-d-glucopyranoside and p-nitrophenyl-β-d-galactopyranoside were acid hydrolyzed and assayed with the Cu-BCS reagent, the amount of sugar released from each of the p-nitrophenyl compounds was found to be equal to the levels of p-nitrophenol in solution. This method is easy to use and with minor modifications can be employed for the quantification of d-glucose and d-galactose in other glycoconjugates.     

Action of 5-thio-D-glucose and its 1-phosphate with hexokinase and phosphoglucomutase.

Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a K_m of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a K_i of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a K_m of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a K_i of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

Molecular and kinetic parameters of sugar transport across the frog choroid plexus

Summary The nature of sugar transport across the blood-cerebrospinal fluid barrier has been investigated using anin vitro preparation of the frog posterior choroid plexus. The permeability of 41 sugars and related compounds was measured by the rapid osmotic procedure described previously. Sugar permeation was found to be stereospecific, inhibited by 1,5-difluoro-2,4-dinitrobenzene, insensitive to anoxia, and independent of the external alkali cation composition. In addition, the transport of a sugar was inhibited by structural analogues. Transport occurred equally well from the ventricular or serosal surface of the tissue, and the rate of transport could be described formally by Michaelis-Menten kinetics. The results were analyzed in terms of the conformation of the sugars in aqueous solution. Sugars which were transported have the d-glucose chair conformation. There is a good correlation between the affinity of the sugar for the transport system and the number of hydroxyl groups attached to the equatorial plane of the ring; d-glucose with five equatorial hydroxyl groups has the greatest affinity. It is concluded that sugar transport across the choroid plexus occurs by facilitated diffusion.     

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°C and 75 mM and 120 mM at 20°C. Reducing the temperature, or raising the loading concentration raises the apparent K_m for net exit. Deoxygenation also reduces the K_m for d-glucose exit from red blood cells loaded initially to 120 mM at 20°C from 32.9 ± 2.3 mM (13) with oxygenated blood to 20.5 ± 1.3 mM (17) (P<0.01). Deoxygenation increases the ratio V_max/K_m from 5.29 ± 0.26 min⁻¹ (13) for oxygenated blood to 7.13 ± 0.29 min⁻¹ (17) for deoxygenated blood (P < 0.001). The counterflow of d-glucose from solutions containing 1 mM ¹⁴C-labelled d-glucose was measured at 2°C and 20°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°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°C and at 2°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.     

Renal sodium-d-glucose cotransport system. Involvement of tyrosine residues in sodium-transporter interaction

Using brush-border membrane vesicles isolated from calf kidney cortex the effect of tyrosine-reactive reagents on sodium-dependent d-glucose transport was investigated. Treatment of the membranes for 60 min with NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), N-acetylimidazole or tetranitromethane decreased d-glucose uptake 50, 70 and 40%, respectively. Tracer exchange experiments revealed that the inhibition of transport is due to a direct modification of the sodium-d-glucose cotransport system. The modification by NBD-Cl decreases the apparent V_max of the transport system with respect to its interaction with sodium. In addition, the rate of inactivation of the transport system by NBD-Cl is reduced in the presence of high concentrations of sodium. The results indicate that tyrosine residues play an essential role in sodium-d-glucose cotransport and are probably involved in the binding and/or transport of sodium by the sodium-d-glucose cotransport system.     

Evidence for negative cooperativity in human erythrocyte sugar transport

1. When d-glucose exchange influx is measure over a wide range of concentrations then two affinity constants (2.27 and 26.0 mM) are evident. This is consistent with a transport model (the allosteric pore model) in which there is negative cooperativity between subunits of the transport protein. 2. The equations for the allosteric pore model interacting with two substrates (or a substrate and an inhibitor) have been derived and have been used to analyse data from exchange inhibition and for mixed infinite-trans uptake experiments. 3. The exchange inhibition of tracer 3-O-methyl-d-glucose, d-xylose and d-fructose uptake by d-glucose also shows evidence for negative cooperativity and for two inhibition constants which are approximately equal to the d-glucose equilibrium exchange affinity constants. 4. The uptake of d-glucose into infinite-transd-glucose or 3-O-methyl-d-glucose gives K_m values of 2.6 and 2.33 mM, respectively. The uptake of 3-O-methyl-d-glucose into infinite-transd-glucose or 3-O-methyl-d-glucose gives K_m values of 6.0 and 4.6 mM, respectively. V values are slightly higher when the internal sugar is 3-O-methyl-d-glucose. 5. In cells that are treated with fluorodinitrobenzene the apparent K_i value for d-glucose inhibition of tracer d-fructose uptake is lowered. It is proposed that this is due to a partially selective effect of FDNB on the internal subunit interface stability constant (the internal pore gate).     

A simple apparatus for performing short-time (1–2 seconds) uptake measurements in small volumes; its application to d-glucose transport studies in brush border vesicles from rabbit jejunum and ileum

An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10–20 μl. Using this technique the kinetic parameters K_m and V of d-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the K_m for d-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Glucose transport in thymocyte plasma-membrane vesicles

Calf-thymocyte membrane vesicles, prepared by hypotonic lysis and homogenization, were isolated by standard centrifugal techniques designed for enrichment of plasma membrane. At 20°C, these vesicles equilibrated with d-glucose and 3-O-methyl-d-glucose more rapidly than with l-glucose. About 25% of the equilibrium d-sugar space (6 μl/mg protein) was very slowly penetrated by l-glucose ( ). The time course of d-sugar accumulation in excess of l-glucose accumulation indicated that this space equilibrated with d-glucose and 3-O-methyl-d-glucose with half-times of approximately 0.2–0.4 min. The remainder of the equilibrium d-sugar space (about 75%) appeared equally accessible to both glucose isomers ( to 5 min). This was confirmed in studies of efflux from preloaded vesicles, where the d-glucose space fell with a short half-time (0.2 min) to the l-glucose space, after which the two isomers exited with the same half-time. Addition of sucrose to increase osmolarity reduced both spaces (specific and non-specific) in a manner which indicated that little if any of the vesicle sugar was bound. This was confirmed by the fact that equilibrium glucose space was independent of glucose concentration and by the fact that vesicles immediately lost their sugar when diluted with water at 0°C. These data indicate the presence of two vesicle types, discriminant and indiscriminant as regards transport of the glucose isomers. Entry of d-glucose into the discriminant (stereospecific) vesicles was temperature sensitive (Q₁₀ > 2), saturable (K_m 2 mM), and was inhibited by phloretin (K_i < 200 μM), N-ethylmaleimide (K_i < 10 mM) and cytochalasin B (K_i < 2 μM), suggesting that these vesicles contain the plasma-membrane glucose carrier. Entry of l- and d-glucose into the indiscriminant vesicles showed none of these properties. The equilibrium-exchange K_m and V were about five times the entry K_m and V, indicating the substrate loading greatly facilitates carrier translocation, at least in the outward direction.     

Specificity of d-glucose transport by the apical membrane of Nereis diversicolor epidermis

(1) The specificity of d-[6-³H]glucose influx by a Na⁺-dependent and phlorizin-sensitive transport system in the apical epidermal membrane of the polychaete worm, Nereis diversicolor, was investigated in vivo. (2) The inhibitory effect of eleven d-glucose analogues on d-[6-³H]glucose influx from a 5 μM external concentration was recorded. The inhibitors (each tested at 5, 50, 500 and 5000 μM) were selected to illuminate the configurational requirements for interaction with the d-glucose transport system. (3) The following compounds were found to be significant inhibitors: methyl α-d-glucoside, methyl β-d-glucoside, d-galactose, $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$, 2-deoxy-d-glucose, d-xylose, myo-inositol, β-d-fructose; the effect was graded according to inhibitor concentration. l-Glucose also inhibited d-glucose influx but to the same extent at all four concentrations tested, suggesting transport site heterogeneity. d-Mannose and l-arabinose did not inhibit influx. (4) The most potent inhibitor, methyl-α-d-glucoside, was itself a substrate, and its transport was inhibited by phlorizin and d-glucose, as well as by substitution of Na⁺ in the incubation medium with Li⁺ or choline⁺. (5) We conclude that the specificity of the Na⁺-dependent d-glucose transporter in the apical epidermal membrane of Nereis is similar to that in the apical membrane of vertebrate small intestinal and proximal tubular epithelium, and in the tapeworm integument.     

Pathways of D-fructose transport in Arthrobacter pyridinolis

Previous work indicated that Arthrobacter pyridinolis can transport d-fructose by either a phosphoenolpyruvate: d-fructose phosphotransferase system or by a respiration-coupled system. The respiration-coupled transport system for d-fructose, which is stimulated by the addition of l-malate, has been characterized in membrane vesicles from d-fructose-grown cells. Such vesicles carry out malate-dependent uptake of d-fructose but not of d-glucose or l-rhamnose, indicating that there is a sugar-specific component to the respiration-coupled transport system. A mutant which is deficient in the d-fructose-specific component was isolated. Vesicles from fructose-glutamate-grown cells of a phosphotransferase-negative strain (AP100) exhibited malate-dependent d-fructose uptake, while phosphoenolpyruvate-dependent uptake was reduced to a small fraction of that seen with vesicles from wild-type cells. Inhibitors of electron transport, carbonyl cyanide m-chlorophenyl hydrazone, 2,4-dinitrophenol and N-ethylmaleimide caused marked inhibition of malate-dependent d-fructose uptake while exerting little or no effect on phosphoenolpyru-vate-dependent transport of the sugar in vesicles from wild-type cells. Activity of a flavin adenine dinucleotide-linked l-malic dehydrogenase was detected in membrane vesicles as well as in whole cells.     

Preparative production of hydroquinone from benzoquinone catalysed by immobilized d-glucose oxidase

Benzoquinone can replace O₂ as an electron acceptor in the oxidation of d-glucose catalysed by A. niger d-glucose oxidase. As a result, a useful chemical, hydroquinone, is formed in nearly 100% yield. A column packed with d-glucose oxidase immobilized onto alumina was operated for two weeks with no measurable decline in its catalytic efficiency and produced more than one hundred grams of hydroquinone.