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At least 1,387 records · Page 77Linked to original sources
PubMed1985

A kinetic analysis of hexose transport in cultured human lymphocytes (IM-9).

3-O-Methyl-D-glucose transport across the plasma membrane of cultured human lymphocytes of the IM-9 line was followed for net entry into sugar-free cells (zero trans entry), net exit into sugar-free medium (zero trans exit) and for equilibration of labelled sugar in cells with the same sugar concentration in the intracellular water as in the medium (equilibrium exchange). The measurements were performed at 37 degrees C (pH 7.4). Equilibrium exchange of 1 mM 3-O-methylglucose (t 1/2 about 7 S) was exponential, suggesting a homogeneous cell suspension. Initial rates of transport showed a Michaelis-Menten dependency on the sugar concentration. The transport system was found to be asymmetric with the following kinetic parameters. Zero trans entry: Km = 2.8 mM, Vmax = 10.7 mM X min-1. Zero trans exit: Km = 9.5 mM, Vmax = 37.9 mM X min-1. Equilibrium exchange: Km = 9.9 mM, Vmax = 44.0 mM X min-1. Finally, the affinity constant for the internal site was measured as approx. 1.2 mM using the infinite cis protocol.

3-O-Methylglucose↗
PubMed1987

Cytochalasin inhibition of hexose transport by platelets.

Previously we described a two-transporter model (T1, T2) for galactose uptake by platelets (Horne, M.K. and Hart, J.S. (1986) Biochim. Biophys. Acta 856, 448-456). In the current work we have sought corroborative evidence for this model by studying the effects of cytochalasins on this transport system. Of the various cytochalasins tested, cytochalasin B was the most potent inhibitor (I) of galactose transport, whereas cytochalasin A was less inhibitory and dihydrocytochalasin B and cytochalasin E had no inhibitory effect. The same order of potency was observed for the inhibition of L-glucose diffusion into platelets. The mechanism of cytochalasin B inhibition was investigated in detail. Inhibition of T1 was competitive and required a higher concentration of cytochalasin B (Ki1 approximately 1.7 microM) than inhibition of T2, which was of a mixed type (Ki2 approximately 0.8 microM). The effect of cytochalasin B on T2 could be accounted for by a membrane alteration which enhanced the affinity of the transporter for galactose while simultaneously preventing passage of the TSI complex into the cell. Since a similar effect on membrane permeability would also explain cytochalasin B inhibition of L-glucose diffusion, it is hypothesized that cytochalasin B binds to a membrane structure shared by T2 and the passage for L-glucose. The differences in cytochalasin B sensitivity and mechanism of inhibition manifested by T1 and T2 support our original hypothesis that galactose is indeed transported by kinetically distinct agencies and suggest that these may be physically distinct as well.

Biological Transport↗
PubMed1987

Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change.

The human erythrocyte sugar transporter has been labelled at its internal site with cytochalasin B and at its outside site by the azidosalicoyl derivative of bis(D-mannose) (ASA-BMPA). The cleavage of the transporter by various proteinases has been studied. Chymotrypsin, subtilisin and V8 proteinase give parallel fragmentation patterns for the two labels down to fragments as small as 7 kDa. Thus the binding sites for the two labels can only be separated by a small span of protein. 2-Nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteines to give a 15 kDa fragment from the two labels. N-Bromosuccinimide (a reagent which preferentially cleaves at tryptophan residues) has revealed differences in fragmentation of the transporter labelled with either cytochalasin B or with ASA-BMPA. A major cleavage site is proposed to occur at tryptophan 186 which leaves a C-terminal fragment containing both labels. A tryptophan cleavage at residue 388 divides the cytochalasin B site and the ASA-BMPA site. A further tryptophan cleavage gives a cytochalasin B labelled 3 kDa fragment probably from residues 388-412. This gives an assignment of the cytochalasin B site as the inside of the hydrophobic span H 10. Since the ASA-BMPA site is probably only 7 kDa from residue 388 and is on the same 15 kDa NTCB fragment as cytochalasin B we assign this to the outside of hydrophobic span H 9. Thermolysin only cleaves the transporter labelled with cytochalasin B and not with ASA-BMPA. A 18 kDa cytochalasin B labelled fragment is formed. This is indicative of a change in conformation of the transporter when an outside ligand is bound such that the inside of the hydrogen bonding transmembrane segments H 7 and H 8 (and containing the proposed thermolysin cleavage site) are withdrawn from the cytosolic surface. Thus it appears that the core of the transporter (including the external and internal sites plus the transmembrane channel) is located between segments H 7 and H 10.

Affinity Labels↗
PubMed1988

Photolabeling of erythrocyte and adipocyte hexose transporters using a benzophenone derivative of bis(D-mannose).

The benzophenone derivative of 1,3-bis(D-mannos-4-yloxy)-2-propylamine (BB-BMPA) has been tested as an exofacial photoaffinity label for the sugar transport systems of human erythrocytes and rat adipocytes. The half-maximal inhibition constants for the reagent are 971 microM in erythrocytes and 536 microM in basal and 254 microM in insulin-treated adipocytes. The photolabelling of erythrocyte membranes is very specific for the 50 kDa transporter peptide and is completely displaced by D-glucose. The exofacial photoaffinity labelling of adipocytes also shows labelling of a 50 kDa transporter peptide, which is displaced by cytochalasin B, but extensive nonspecific labelling of a 75 kDa plasma membrane peptide occurs. The transporter is labelled in insulin-treated cells but not in basal cells which indicates that this in situ labelling technique selectively reveals only those transporters that visit and are active in the plasma membrane during the labelling period. This also indicates that in basal cells transporters do not turn over rapidly. Subcellular redistribution of transporters after the labelling period has been studied. Following incubation and washing at 37 degrees C in the presence of insulin, 30% of the transporters photolabelled at the plasma membrane are internalised and are found in the light microsome fraction of the cell. The proportion of transporter that is observed to be internalised is much greater than can be accounted for by a contamination of the light microsome fraction by plasma membrane. The labelled 50 kDa transporter peptide in the light microsomes is enriched when compared with the carry-over of the 75 kDa nonspecifically labelled plasma membrane peptide. Thus we have obtained direct evidence for transporter translocation.

Adipose Tissue↗
PubMed1995

Use of hexose transport mutants to examine the expression and properties of the rat myoblast GLUT 1 transport process.

Rat L6 myoblasts were recently shown to possess the GLUT 1, 3 and 4 transporters, and not the GLUT 2 isoform [1]. This investigation examined the expression and properties of the GLUT 1 isoform. GLUT 1 transcript level was significantly reduced in cells grown at high densities and during myogenic differentiation. A comparison of the GLUT 1 and 4 transcript levels in myogenesis-competent and impaired cells revealed an inverse relationship between these two isoforms. This relationship was confirmed by studies using two independent spontaneous GLUT 3- GLUT 4- mutants, M1 and M3. These mutants possessed very high level of the GLUT 1 isoform, but negligible amount of the GLUT 3 and 4 isoforms. GLUT 1 expression was also subject to positive regulation. Glucose starvation was found to increase not only the levels of the GLUT 1 transcript and transporter, but also the intrinsic activity of the GLUT 1 transporter. Studies with M1 and M3 mutants revealed that the GLUT 1 transporter was not functional in glucose-grown cells, even though it was present at a very high level in the plasma membrane. This transporter became functional when cells were starved for glucose. The functional GLUT 1 transporter had an apparent Km value of around 0.9 mM, and was sensitive to cytochalasin B, phloretin, phlorizin and pCMBS.

3-O-Methylglucose↗
PubMed1994

Re-examination of hexose exchanges using rat erythrocytes: evidence inconsistent with a one-site sequential exchange model, but consistent with a two-site simultaneous exchange model.

(1). The kinetic parameters of zero-trans net uptake and infinite-trans uptake of 3-O-methyl-D-glucoside, 2-deoxy-D-glucose and D-mannose into rat red cells at 24 degrees C were measured after taking account of the linear diffusion components of flux. (2). Zero-trans exists of 3-O-methyl-D-glucoside and D-mannose from rat cells were also measured. (3). After correction for linear flux via non-specific routes, the Vmax of zero-trans uptake of 3-O-methyl-D-glucoside was significantly higher, (1.25 +/- 0.06 mumol (10 min)-1 (ml cell water)-1) than the corresponding parameters of mannose or 2-deoxy-D-glucose, (0.33 +/- 0.01 and 0.39 +/- 0.01 mumol(10 min)-1 (ml cell water)-1, respectively; P < 0.001). (4). After correction for linear flux via non-specific uptake routes, the Vmax of zero-trans exit of 3-O-methyl-D-glucoside is significantly higher (1.70 +/- 0.1 mumol (10 min)-1 (ml cell water)-1) than the corresponding value for mannose exit flux, (1.10 +/- 0.1 mumol (10 min)-1 (ml cell water)-1; P < 0.001). (5). The acceleration ratio, i.e., the ratio of infinite-trans influx Vmax/zero-trans influx Vmax of mannose by mannose (9.12 +/- 0.03) is significantly higher than that of 3-O-methyl-D-glucose by 3-O-methyl-D-glucose (2.77 +/- 0.14)(P < 0.001). (6). The one-site simple carrier model of glucose transport in which sugar exchange is viewed as a sequential process, predicts that the acceleration ratio of the more rapidly moving sugar 3-O-methyl-D-glucose by 3-O-methyl-D-glucose should be greater than that of the slower sugar, mannose by mannose. Hence, the observed findings are inconsistent with the one-site model, but confirm the earlier disputed studies of Miller, D.M. (1968; Biophys. J. 8, 1329-1338). (7). A two-site model, in which sugar exchange is considered as a simultaneous process, predicts that the acceleration ratio of mannose influx by mannose should be higher than for 3-O-methyl-D-glucose by 3-O-methyl-D-glucose. The data are, therefore, consistent with a two-site model.

Animals↗
PubMed1995

Preferential association of membrane phospholipids with the human erythrocyte hexose transporter.

This study reports the results of an investigation to determine to what extent the influence of membrane lipids on the human erythrocyte sugar transporter protein activity (Caruthers, A. and Melchior, D.L. (1988) Annu. Rev. Physiol. 50, 257-271) is related to lipid/protein associations in the membrane bilayer. Differential scanning calorimetry was carried out on the human erythrocyte transport protein reconstituted into artificial bilayers formed from preselected lipids. it was found that the transport protein displays a preferential and in some cases strongly preferential affinity for specific lipid types. This association is a function of lipid head group, backbone and hydrocarbon chain length. It appears that the affinity of the transport protein for various lipids can correlate with the lipid's ability to influence transporter activity. This study further suggests that certain lipids (in this case sphingomyelin) can induce an oligomeric association of HEST monomers in the bilayer.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1976

Stabilization of glucosaminephosphate synthase from rat liver by hexose 6-phosphates. Properties and interconversion of two molecular forms.

Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37 degrees C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methyl-glyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography. During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore intercovertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A. These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase.

Animals↗
PubMed1991

Feeding rats a diet enriched with saturated fatty acids prevents the inhibitory effects of acute and chronic ethanol exposure on the in vitro uptake of hexoses and lipids.

Chow-fed rats were given 15% ethanol in their drinking water for 4 weeks, and then for the next 2 weeks of ethanol exposure they were fed isocaloric semisynthetic diets enriched in either saturated (S) or polyunsaturated (P, linoleic acid) fats. Food intake was lower in ethanol-fed (ETH) than in control (C) rats, but the average body weight gain was similar in ETH and C fed S or P. Intestinal dry weight and the percentage of the intestinal wall comprised of mucosa were more than 2-fold higher in ETH than C fed P, whereas these values were 50% lower in ETH than C fed S. The in vitro jejunal uptake of glucose and galactose was higher in ETH than C fed S, whereas the converse was true when feeding P. These effects were due to differences in the values of the maximal transport rate (Vmax), the Michaelis constant (Km), and the contribution of passive permeation. The relative permeability of the intestine to lipids was unchanged by giving ethanol or by feeding S or P, but the individual rates of uptake of most medium- and long-chain fatty acids and cholesterol were lower in ETH fed P as compared with S. In a second series of studies the acute effect of ethanol exposure was examined: animals were fed S or P for 2 weeks and the intestine was then removed: when 5% ethanol was added directly to the test solutions, there was lower in vitro jejunal and ileal uptake of glucose and higher jejunal uptake of 18:2 when rats were previously fed P, but not in those fed S. In summary; (1) feeding an isocaloric polyunsaturated fatty acid diet has a trophic effect on the intestinal mucosa of animals chronically drinking ethanol; and (2) feeding rats a diet enriched with saturated fatty acids prevents the inhibitory effects of acute and chronic ethanol exposure on the in vitro jejunal uptake of glucose, galactose and lipids observed in animals fed a polyunsaturated diet. Thus, the effect of chronic consumption of ethanol on the active and passive jejunal uptake of nutrients is influenced by the type of lipids in the animal's diet.

Alcoholism↗