Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding

Biochemistry ◽  
1991 ◽  
Vol 30 (49) ◽  
pp. 11546-11553 ◽  
Author(s):  
Anthony P. J. King ◽  
Ping Kaung Ku Tai ◽  
Christin Carter-Su
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Sugar Binding

scholarly journals Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter

1992 ◽  
Vol 281 (1) ◽  
pp. 103-106 ◽  
Author(s):  
H Nishimura ◽  
H Kuzuya ◽  
A Kosaki ◽  
M Okamoto ◽  
M Okamoto ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Conformational Changes ◽  
Human Erythrocyte ◽  
Binding Sites ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Tertiary Structure ◽  
Erythrocyte Membranes ◽  
C Terminus ◽  
Low Concentrations

Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane.

scholarly journals Glycation of the human erythrocyte glucose transporter in vitro and its functional consequences

1990 ◽  
Vol 268 (3) ◽  
pp. 661-667 ◽  
Author(s):  
P J Bilan ◽  
A Klip
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Competitive Inhibitor ◽  
Human Erythrocyte Membrane ◽  
Functional Consequences ◽  
High Concentrations ◽  
Erythrocyte Membrane Proteins

Glycation of human erythrocyte membrane proteins was induced by incubation in vitro with high concentrations (80 mM or 200 mM) of D-glucose for 3 or 6 days. The extent of glycation was quantified from the covalent incorporation of 3H by reduction of the glucose glycation products with NaB3H4. For membranes incubated for 3 days with 80 mM-D-glucose, glycation in vitro of Band 4.5 (containing the glucose transporter) was equivalent to 0.11 mol of glucose/mol of glucose transporter, compared with 3H labelling in 3-day-incubated control membranes of 0.055 mol of glucose/mol of glucose transporter. In membranes incubated for 6 days with 200 mM-D-glucose, glycation increased to 0.21 mol of glucose/mol of glucose transporter, whereas the controls without glucose had 0.11 mol of glucose/mol of glucose transporter. Glycation in vitro was accompanied by a fall in the Bmax of binding of [3H]cytochalasin B (a competitive inhibitor of glucose transport), without any change in the binding affinity. The data suggest that glycated glucose transporters have decreased ability to bind cytochalasin B. It is proposed that glycation can alter glucose transporter activity.

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.

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 Proteolytic and chemical dissection of the human erythrocyte glucose transporter

1984 ◽  
Vol 221 (1) ◽  
pp. 179-188 ◽  
Author(s):  
M T Cairns ◽  
D A Elliot ◽  
P R Scudder ◽  
S A Baldwin
Keyword(s):  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Diffuse Band ◽  
Cytoplasmic Surface ◽  
Cytoplasmic Face ◽  
Membrane Bound ◽  
Beta Galactosidase

Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.

Binding of cytochalasin B to human erythrocyte glucose transporter

Biochemistry ◽  
1980 ◽  
Vol 19 (23) ◽  
pp. 5417-5420 ◽  
Author(s):  
David C. Sogin ◽  
Peter C. Hinkle
Keyword(s):  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B

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).

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