Characterization of cytochalasin B photoincorporation into the human erythrocyte D-glucose transporter and F-actin

Biochemistry ◽  
1983 ◽  
Vol 22 (11) ◽  
pp. 2750-2756 ◽  
Author(s):  
Michael F. Shanahan
Keyword(s):  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B

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 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.

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.

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium

Parasitology ◽  
1998 ◽  
Vol 117 (6) ◽  
pp. 579-588 ◽  
Author(s):  
D. RODRÍGUEZ-CONTRERAS ◽  
P. J. SKELLY ◽  
A. LANDA ◽  
C. B. SHOEMAKER ◽  
J. P. LACLETTE
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Functional Characterization ◽  
Taenia Solium ◽  
Cdna Clones ◽  
Facilitated Diffusion ◽  
Porcine Cysticercosis ◽  
Isolation And Characterization ◽  
Hexose Uptake

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers d-glucose and d-mannose but not by l-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

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 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
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