scholarly journals Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein

1988 ◽  
Vol 256 (2) ◽  
pp. 421-427 ◽  
Author(s):  
A F Gibbs ◽  
D Chapman ◽  
S A Baldwin
Keyword(s):  
Conformational Change ◽  
Glucose Transport ◽  
Conformational Changes ◽  
Steric Hindrance ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytoplasmic Surface ◽  
Tryptic Cleavage ◽  
Hydrophilic Region ◽  
Glucose Transport Protein

Tryptic digestion has been used to investigate the conformational changes associated with substrate translocation by the human erythrocyte glucose transporter. The effects of substrates and inhibitors of transport on the rates of tryptic cleavage at the cytoplasmic surface of the membrane have confirmed previous observations that this protein can adopt at least two conformations. In the presence of phloretin or 4,6-O-ethylidene-D-glucose, the rate of cleavage is slowed. Because these inhibitors bind preferentially at the extracellular surface of the transporter, their effects must result from a conformational change rather than from steric hindrance. A conformational change must also be responsible for the effect of the physiological substrate D-glucose, which is to increase the rate of cleavage. The regions of the protein involved in the conformational changes include both of the large cytoplasmic regions that are cleaved by trypsin: these are the central hydrophilic region of the sequence (residues 213-269) and the hydrophilic C-terminal region (residues 457-492).

scholarly journals Ligand-induced conformational changes modify proteolytic cleavage of the adipocyte insulin-sensitive glucose transporter

1993 ◽  
Vol 295 (1) ◽  
pp. 183-188 ◽  
Author(s):  
Y Yano ◽  
J M May
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Proteolytic Cleavage ◽  
Specific Antiserum ◽  
Tryptic Cleavage ◽  
Glut1 Protein ◽  
Membrane Bound ◽  
Glucose Transporter Glut4 ◽  
Glucose Transporter Glut1

The transport conformation of the human erythrocyte glucose transporter (GLUT1) modifies rates of proteolytic cleavage of this protein by a variety of enzymes. We investigated the effects of ligand-induced conformational change on the susceptibility to enzymic cleavage of the insulin-sensitive rat adipocyte glucose transporter (GLUT4). A GLUT4-enriched slow sedimenting microsomal fraction was prepared from basal adipocytes and subjected to PAGE and immunoblotting. The GLUT4 protein was detected in these immunoblots with a C-terminal-specific antiserum as an M(r)-46,000-50,000 doublet. GLUT1 protein was not detected by a GLUT1-specific antiserum in these membranes. Tryptic digestion caused loss of the GLUT4 signal in immunoblots in a time- and concentration-dependent fashion. Low-M(r) membrane-bound fragments were not observed in electrophoretic gels, whether detection was attempted by immunoblotting or by counting radioactivity in gel slices following photolabelling with [3H]cytochalasin B. Transport-specific ligands known to induce an outward-facing conformation in the human erythrocyte GLUT1 protein retarded cleavage of the GLUT4 protein by submaximal concentrations of trypsin, whereas ligands known to induce an inward-facing conformation increased the extent of cleavage. The transported substrate D-glucose retarded tryptic cleavage of GLUT4. This result contrasts with the known behaviour of GLUT1, in which D-glucose accelerates cleavage. Cleavage of GLUT4 by thermolysin was also retarded by the outward-binding analogue 4,6-O-ethylidene glucose. These results show that the conformational sensitivity to proteolysis of GLUT4 mirrors that of GLUT1, except that the glucose-loaded GLUT4 has a different steady-state configuration, which may reflect underlying kinetic differences between the two proteins.

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.

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.

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity

1990 ◽  
Vol 1030 (1) ◽  
pp. 60-64 ◽  
Author(s):  
Jean-Paul Feugeas ◽  
Dominique Néel ◽  
AndréA. Pavia ◽  
Antoun Laham ◽  
Yves Goussault ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Transport Activity
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