scholarly journals Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose

1990 ◽  
Vol 269 (3) ◽  
pp. 615-622 ◽  
Author(s):  
A E Clark ◽  
G D Holman
Keyword(s):  
Transport System ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Erythrocyte Membranes ◽  
Trypsin Treatment ◽  
Intact Cells ◽  
Tryptic Fragment ◽  
System A ◽  
Glucose Transport System ◽  
Peptide Antibody

The synthesis of 2-N-[4-(1′-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.

Fructose transport byHaloferax volcanii

1995 ◽  
Vol 41 (3) ◽  
pp. 241-246 ◽  
Author(s):  
Jin-Ichiro Takano ◽  
Kouichi Kaidoh ◽  
Naoki Kamo
Keyword(s):  
Kinetic Analysis ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Transport System ◽  
Driving Force ◽  
Glucose Transporter ◽  
Potential Gradient ◽  
Haloferax Volcanii ◽  
Intact Cells ◽  
Glucose Transport System

Uptake of fructose by intact cells of Haloferax volcanii, one of the sugar-utilizing halobacteria, was examined with the following results. (i) The fructose transporter was inducible, (ii) Kinetic analysis showed a Ktof 0.37 μM and a Vmaxof 4.61 nmol∙mg protein−1∙min−1. For a glucose transport system in this bacterium, Ktwas 12.5 μM, showing that the fructose transporter had a much larger affinity for a substrate than the glucose transporter, (iii) No uptake was observed by the envelope vesicles, (iv) Phloridzin and phloretin inhibited fructose transport, although a relatively higher concentration was required than that needed to inhibit glucose uptake, (v) The driving force for fructose transport was a Na+electrochemical potential gradient, (vi) Glucose and fructose were interchangeable and this conversion led to the expression of the glucose transporter even when fructose was used as the sole carbohydrate, and vice versa.Key words: glucose uptake, symport with Na+, phloridzin, phloretin, Haloferax volcanii.

scholarly journals Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system

1975 ◽  
Vol 145 (3) ◽  
pp. 417-429 ◽  
Author(s):  
J E Barnett ◽  
G D Holman ◽  
R A Chalkley ◽  
K A Munday
Keyword(s):  
Glucose Transport ◽  
Transport System ◽  
Human Erythrocyte ◽  
Partition Coefficients ◽  
External Medium ◽  
Sugar Transport ◽  
Conformational States ◽  
Glucose Transport System ◽  
Oil Water

6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.

Sulfhydryl substituents of the human erythrocyte hexose transport mechanism

1986 ◽  
Vol 250 (6) ◽  
pp. C853-C860 ◽  
Author(s):  
R. E. Abbott ◽  
D. Schachter ◽  
E. R. Batt ◽  
M. Flamm
Keyword(s):  
Human Erythrocyte ◽  
Transport Mechanism ◽  
Cytochalasin B ◽  
Erythrocyte Membranes ◽  
Type I ◽  
Type Ii ◽  
Hexose Transport ◽  
Intact Cells ◽  
Type Iii ◽  
Human Erythrocyte Membranes

Sulfhydryl substituents of the hexose transport mechanism of human erythrocyte membranes were studied with membrane-impermeant and -permeant maleimide derivatives. Three sulfhydryl classes have been identified on the basis of their reactivity toward the reagents and their effects on the transport mechanism. Type I sulfhydryl is located at the outer (exofacial) surface of the membrane and bound covalently on treatment of intact cells with the membrane-impermeant glutathione-maleimide. This sulfhydryl is required for the transport, and it is protected from alkylation, i.e., its reactivity toward maleimides is decreased by the presence of D-glucose or cytochalasin B. Type II sulfhydryl is also required for the transport, but it differs from type I in that D-glucose (but not cytochalasin B) increases the reactivity toward maleimides. Further, it is located at the endofacial surface of the membrane, since reaction with glutathione-maleimide occurs only in leaky ghosts and not in intact cells. Alkylation by glutathione-maleimide of type I and type II sulfhydryls increases the half-saturation for the binding of D-glucose to erythrocyte membranes. In contrast, inactivation of type III sulfhydryls by N-ethylmaleimide or dipyridyl disulfide decreases the half-saturation concentration for the binding of D-glucose and other transported hexoses to the membranes; nontransported sugars are not affected similarly. Type III sulfhydryl is not inactivated by the polar reagent glutathione-maleimide and is probably located in a nonpolar domain of the transport mechanism. Inactivation of either type I or II sulfhydryls decreases or eliminates the flux asymmetry of the hexose transport mechanism.

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

1988 ◽  
Vol 66 (10) ◽  
pp. 1126-1133 ◽  
Author(s):  
Elena Burdett ◽  
Amira Klip
Keyword(s):  
Metabolic Regulation ◽  
Glucose Transporter ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Glucose Transporters ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Western Blots ◽  
Intact Cells ◽  
Human Erythrocyte Membranes

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55 000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze–thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters. It is concluded that the antibody may be reacting with exofacial regions of the glucose transporter in intact cells. This suggests that the antibodies may be used to detect relative changes in glucose transporter number on the cell surface.

FUNCTIONAL AND MOLECULAR CHARACTERISTICS OF A PRIMITIVE VERTEBRATE GLUCOSE TRANSPORTER: STUDIES OF GLUCOSE TRANSPORT BY ERYTHROCYTES FROM THE PACIFIC HAGFISH (EPTATRETUS STOUTI)

1994 ◽  
Vol 186 (1) ◽  
pp. 23-41 ◽  
Author(s):  
J. D. Young ◽  
Y. Syn ◽  
C. M. Tse ◽  
A. Davies ◽  
S. A. Baldwin
Keyword(s):  
Glucose Transport ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Sequence Similarity ◽  
Erythrocyte Membranes ◽  
Molecular Characteristics ◽  
Relative Molecular Mass ◽  
Amino Acid Sequence Homology ◽  
The Pacific

The characteristics of glucose transport were investigated in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti) Lockington. Transport of glucose by intact hagfish erythrocytes and by phospholipid vesicles reconstituted with n-octylglucoside extract of hagfish erythrocyte membranes was rapid and mediated by a saturable stereospecific mechanism sensitive to inhibition by cytochalasin B. Covalent photoaffinity labelling experiments with [3H]cytochalasin B identified the hagfish glucose transporter on SDS/polyacrylamide gels as a protein with an apparent average Mr of 55 000. Amino acid sequence homology between the hagfish and human erythrocyte glucose transporters (GLUT 1) was investigated in immunoblotting experiments using a panel of 12 different antipeptide antisera and affinity-purified antibodies raised against cytoplasmic extramembranous regions of the human transporter, and with an antibody to the intact purified human protein. The latter antibody labelled a component in the membrane with the same apparent Mr as cytochalasin B. Two affinity-purified antipeptide antibodies, corresponding to residues 240–255 and 450–467 of the human erythrocyte transporter, also labelled a component in the membrane with this relative molecular mass, demonstrating localised sequence similarity between the polypeptides of the two species within the central cytoplasmic loop and within the cytoplasmic C-terminal region. Glucose transport by hagfish erythrocytes was not coupled to the movement of protons.

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