scholarly journals Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus

1992 ◽  
Vol 283 (3) ◽  
pp. 643-646 ◽  
Author(s):  
C K Yi ◽  
B M Charalambous ◽  
V C Emery ◽  
S A Baldwin
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Transport Inhibitor ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

The human erythrocyte-type glucose transporter (GLUT1) has been abundantly expressed in insect cells by using a recombinant baculovirus. At 4 days after infection with the virus, the insect cell-surface and intracellular membranes were found to contain greater than 200 pmol of D-glucose-sensitive binding sites for the transport inhibitor cytochalasin B per mg of protein. The characteristics of binding were identical with those of the erythrocyte transporter, although the two proteins differed substantially in apparent Mr, probably as a result of glycosylation differences.

scholarly journals Domain assembly of the GLUT1 glucose transporter

1994 ◽  
Vol 300 (2) ◽  
pp. 291-294 ◽  
Author(s):  
D L Cope ◽  
G D Holman ◽  
S A Baldwin ◽  
A J Wolstenholme
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Insect Cells ◽  
Full Length ◽  
Stable Complex ◽  
C And N ◽  
Terminal Amino ◽  
Glut1 Glucose Transporter

A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation.

d-Glucose, forskolin and cytochalasin B affinities for the glucose transporter Glut1

1997 ◽  
Vol 776 (1) ◽  
pp. 81-86 ◽  
Author(s):  
Lili Lu ◽  
Andreas Lundqvist ◽  
Cheng-Ming Zeng ◽  
Christine Lagerquist ◽  
Per Lundahl
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

scholarly journals Identification and characterization of the glucose-transport protein of the bovine blood/brain barrier

1987 ◽  
Vol 247 (1) ◽  
pp. 101-108 ◽  
Author(s):  
M A Kasanicki ◽  
M T Cairns ◽  
A Davies ◽  
R M Gardiner ◽  
S A Baldwin
Keyword(s):  
Cerebral Cortex ◽  
Glucose Transport ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Broad Band ◽  
Transport Protein ◽  
Western Blots ◽  
Glucose Transport Protein

The glucose-transport protein from bovine cerebral-cortex microvessels has been identified and characterized by virtue of its ability to bind the ligand [4-3H]cytochalasin B. Microvessel membranes were found to contain a single set of glucose-inhibitable high-affinity cytochalasin B-binding sites [113 +/- 16 (S.E.M.) pmol/mg of membrane protein], with an association constant of 6.8 +/- 1.8 (S.E.M.) micron-1. D-Glucose inhibited the binding to these sites with a Ki of 31 mM. The transport protein was identified by photoaffinity labelling with [4-3H]cytochalasin B and was found to migrate as a broad band of apparent Mr 55,000 on SDS/polyacrylamide gels. Labelling was inhibited by D-glucose, but not by L-glucose. Treatment with endoglycosidase F yielded a sharper band of apparent Mr 46,000, indicating that the transport protein is glycosylated. However, in contrast with the human erythrocyte glucose transporter, digestion with endo-beta-galactosidase had little effect on the electrophoretic mobility of the microvessel protein. Tryptic digestion of the photolabelled protein yielded a radioactive fragment of apparent Mr 18,000, similar to that of the fragment produced by digestion of the labelled human erythrocyte glucose transporter. In addition, a protein of Mr identical with that of the photolabelled transporter was labelled on Western blots of microvessel membranes by antisera raised against the intact erythrocyte transporter and against synthetic peptides corresponding to its N- and C-terminal regions. It is concluded that the glucose-transport protein of bovine cerebral-cortex microvessel endothelial cells shows structural homology with the human erythrocyte glucose transporter.

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