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

1987 ◽  
Vol 15 (6) ◽  
pp. 1128-1129
Author(s):  
ANGELA F. GIBBS ◽  
DENNIS CHAPMAN ◽  
STEPHEN A. BALDWIN
Keyword(s):  
Glucose Transport ◽  
Conformational Changes ◽  
Human Erythrocyte ◽  
Transport Protein ◽  
Glucose Transport Protein

Properties of the Human Erythrocyte Glucose Transport Protein Are Determined by Cellular Context†

Biochemistry ◽  
2005 ◽  
Vol 44 (15) ◽  
pp. 5606-5616 ◽  
Author(s):  
Kara B. Levine ◽  
Trista K. Robichaud ◽  
Stephanie Hamill ◽  
Lisa A. Sultzman ◽  
Anthony Carruthers
Keyword(s):  
Glucose Transport ◽  
Human Erythrocyte ◽  
Transport Protein ◽  
Cellular Context ◽  
Glucose Transport Protein

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 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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