Affinity For D-glucose Research Articles

Glucose Transport in Human Red Blood Cells

The penetration of D-[14C] glucose into human red blood cells (RBCs) features kinetic parameters which are readily distinguishable from passive permeation. It would be expected to require activation energy above 80 kJ/mol for permeation of glucose with five hydroxyls capable of forming hydrogen bonds, but the measured activation energy is approximately 16 kJ/mol. As a consequence, glucose permeates RBC membrane about five orders of magnitude faster than would be expected for passive permeation. Glucose transporter protein 1, or GLUT1 and SGLT1, present in all human tissues, but especially in RBCs. It is also anchored in the protective sheet of flat cells that line up the blood vessels of the brain. GLUT1 has a strong affinity for glucose and it ensures that both RBCs and the brain receive appropriate levels of glucose that they need to be able to function. The brain consumes ~120g of glucose per day; the blood glucose level in a typical person 80mg/100ml. The binding site of glucose faces intracellular and extracellular of the membrane alternately when it is loaded by a glucose. The transport is accomplished by conformational changes within GLUT1 , and not by rotation of the whole single long polypeptide chain (55kD, ~500 residues) with the presence of 12 trans membrane α-helices segments. The super family of related GLUT sugar transporters comprises 14 identified isoforms in the human genome, all adopting a 12-membrane–spanning domain structure that delineate 6 extracellular loops .The erythrocyte glucose transporter GLUT1 has an ~10-fold-lower affinity for D-glucose, Km ≈ 10–15 mM, at the inside face for net export than on the outside (Km = 1–2 mM) for net import of glucose (zero-trans net flux) at 24°C , pertaining a liganded consequential asymmetric transporter.

Characterization of the lectin purified fromCanavalia ensiformis shoots

Lectin is a cell-agglutinating and carbohydrate-binding protein present in many plants. The lectin ofCanavalia ensiformis shoot with specific affinity ford-glucose was purified by affinity chromatography using Sephadex G-100, and some of its biochemical characterizations were studied. Lectin was purified 8.87-fold and exhibited final specific activity of 225.74 units/mg protein with a 2.3% yield. SDS-PAGE analysis demonstrated that the purified shoot lectin exists as a tetramer of 102 kD, composed of two subunits with molecular weight of 29 and 22 kD. The purified lectin was observed to agglutinate rabbit blood cell. The optimal temperature for the activity of this lectin was 40°C, and this lectin was relatively stable to heat with the highest activity at 50∼60°C. The maximal activity was observed at pH 7.2.

Substitution of tyrosine 293 of GLUT1 locks the transporter into an outward facing conformation

Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.

Sodium-induced conformational changes in the glucose transporter of intestinal brush borders.

Using brush-border membranes prepared from rabbit small intestine by Ca2+ precipitation and KSCN treatment, we have studied the kinetics and conformational changes of the glucose carrier. Na+ behaves as a competitive activator of glucose transport under zero-trans conditions. Phenyl isothiocyanate and fluorescein isothiocyanate (FITC) inhibit Na+-dependent transport in an irreversible but substrate-protectable manner. Vesicles pretreated with phenyl isothiocyanate in the presence of substrates were then selectively labeled at the glucose carrier with FITC. Competition experiments with Na+ and phlorizin or glucose indicated that FITC binds to the glucose site on the carrier. Carrier-bound FITC displays a saturable quenching of fluorescence in the presence of Na+. The K0.5 of the Na+-specific quench is 25 mM, which is similar to the apparent Km for Na+ activation of glucose transport. Two tyrosine group-specific reagents, N-acetylimidazole and tetranitromethane, inhibit glucose uptake and fluorescent quenching in a Na+-protectable fashion. FITC labeled a 75-kilodalton peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a substrate-sensitive manner. We conclude that Na+ binds to the glucose symporter of intestinal brush borders, a 75-kilodalton peptide, and this induces a rapid conformation change in the transporter which increases its affinity for D-glucose.

The Binding of d-Glucose to the Isolated Luminal Membrane of the Renal Proximal Tubule

Abstract The specific binding of d-glucose to the isolated luminal cell membrane of the renal proximal tubule, the brush border, was studied. The initial interaction of d-glucose with this membrane was rapid; saturable; dependent on the concentrations of the sugar and the membrane; reversible; selectively influenced by the ionic environment; sensitive to temperature and pH; inhibited by phloridzin, phloretin, and sulfhydryl reagents; and stereospecific. Examination of the relationship between the concentration of d-glucose and the binding of the sugar indicated the presence of two distinct classes of receptor sites for d-glucose in the brush border membrane. One system, having a high affinity class of binding sites, was saturated at about 1 µm, half-saturated at 0.67 µm, and 1.4 x 10-3 nmoles of d-glucose interacted with 1 mg of membrane protein. The other system, having a low affinity class of binding sites, required 50 mm for saturation, and had an affinity for d-glucose at least four-orders of magnitude less than that of the high affinity receptor sites. The time courses of binding of d-glucose in the high and low affinity regions were similar, in both cases approximately 50% of the maximal bindings occurred in the 1st min of incubation and steady state levels were achieved in about 3 min. The specific bindings of d-glucose at both receptor sites were completely reversible. Neither system of binding required Na+. The initial interaction of d-glucose with the brush border membranes was inhibited by Tris ions, but Li+ and choline ions substituted for Na+. Binding of d-glucose to the brush border membrane was markedly dependent on the presence of Ca2+. Binding at the high affinity classes of sites was more sensitive to the removal of Ca2+ than was the binding at the low affinity classes. The deletion of Mg2+ caused a moderate decrease in the binding at both affinity regions. High concentrations of phosphate inhibited binding at the low affinity classes of binding sites. Glutamine was also inhibitory. Phloridzin was found to be a potent inhibitor of the interaction of d-glucose with the receptors on the renal brush border membrane. Kinetics typical of competitive inhibition were observed at the high affinity region of d-glucose binding. The apparent Ki was 2 x 10-4 m. The kinetics of inhibition at the low affinity classes of binding sites were not consistent with competitive inhibition. The inhibition by phloretin at the high affinity sites was noncompetitive. The sulfhydryl reagents, N-ethylmaleimide and mersalyl, blocked the binding of d-glucose at both the high and low affinity sites. The binding of d-glucose was found to differ from that of l-glucose. Neither the binding of d- or l-glucose was affected by the presence of the other stereoisomer. When incubated together, the total sugar bound was the sum of the d-glucose bound alone plus the l-glucose bound alone. The interaction of l-glucose was insensitive to phloridzin. d-Galactose, a competitive inhibitor of d-glucose binding, did not influence the binding of l-glucose. l-Glucose, in contrast to d-glucose, did not reverse the binding of d-glucose. The l-glucose sequestered by the luminal membranes was not releasable by d- or l-glucose. These notable differences between the interactions of d- and l-glucose with the brush border membranes strongly indicated that the two stereoisomers did not share the same carrier transport system on the proximal tubule luminal membrane. It is proposed that the binding of d-glucose to the isolated brush border membranes, as characterized in this report, represents the initial reaction in the active transport of the sugar by the renal tubule.