uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Cytochalasin B-binding and transport properties of the Glut1 human red cell glucose transporter
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2000 (English)In: Recent research developments in bioenergetics Vol. 1 (2000) Part II, Trivandrumc: Transworld Research Network , 2000, Vol. 1, 117-129 p.Chapter in book (Refereed)
Abstract [en]

The mechanism by which the D-glucose transporter Glut1 facilitates transport of monosaccharides, dehydroascorbic acid and possibly nicotinamide across membranes toward lower chemical activity is thought to involve binding of the substrate at the internal or external face of the protein followed by a thermally triggered conformational change that allows release of the substrate from a binding site exposed at the opposite side of the protein. In the presumably tetrameric transporter in the cell membrane, pairs of identical subunits may work in concert. In each dimer, one of the subunits may expose an external substrate-binding site and the other an internal site at a given moment (State 1). Only the internal site binds the inhibitor cytochalasin B. Cytochalasin B-binding analyses and transport data support this mechanism [Hamill, S., Cloherty, E.K., and Carruthers, A. 1999, Biochemistry, 38, 16974]. Both alkaline disulfide reduction of Glut1 in the cell membrane and polylysine-coating of red cells convert Glut1 to a functionally monomeric state that offers a cytochalasin B-site on every subunit (State 2). Cysteine-scanning mutagenesis experiments with transport and inhibition analyses indicate that the proposed transmembrane segments 5 and 7 are amphipathic α-helices that probably line the glucose permeation pathway. The future determination of the 3D structures of sugar transporters may elucidate the transport mechanism and subunit interactions.

Place, publisher, year, edition, pages
Trivandrumc: Transworld Research Network , 2000. Vol. 1, 117-129 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90675ISBN: 81-86846-59-X (print)OAI: oai:DiVA.org:uu-90675DiVA: diva2:163119
Available from: 2003-09-04 Created: 2003-09-04 Last updated: 2013-06-12Bibliographically approved
In thesis
1. Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter
Open this publication in new window or tab >>Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.

Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 50 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 868
Keyword
Biochemistry, Affinity, Aromatic amino acids, Binding, Biomembrane, Biotin, Chromatography, Cytochalasin B, Dihydrocytochalasin B, Dissociation constant, Drug absorption, Ethanol, Equilibrium, Glucose, GLUT1, Immobilization, Immobilized biomembrane affinity chromatography, Immobilized liposome chromatography, Interaction, Liposome, Membrane protein, Membrane vesicle, Partitioning, Phospholipid bilayer, Proteoliposome, Quantitative, Red blood cell, Specific, Streptavidin, Tyrosine, Tryptophan, Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-3525 (URN)91-554-5692-8 (ISBN)
Public defence
2003-09-26, B:41, BMC, Husarvägen 3, 13:15
Opponent
Supervisors
Available from: 2003-09-04 Created: 2003-09-04 Last updated: 2013-06-10Bibliographically approved
2. Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
Open this publication in new window or tab >>Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction.

Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo.

Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.

An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 43 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 755
Keyword
Biochemistry, Affinity, Binding, Biomembrane, Biotin, Chromatography, Cytochalasin B, Dissociation constant, Drug absorption, Equilibrium, Glucose, GLUT1, Immobilization, Immobilized biomembrane affinity chromatography, Immobilized liposome chromatography, Interaction, Liposome, Membrane protein, Membrane vesicle, Partitioning, Phospholipid bilayer, Proteoliposome, Quantitative, Red blood cell, Solute, Specific, Streptavidin, Wheat germ agglutinin, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-2668 (URN)91-554-5413-5 (ISBN)
Public defence
2002-10-25, lecture hall B41, Biomedical Center, Uppsala, 10:15
Opponent
Available from: 2002-10-03 Created: 2002-10-03 Last updated: 2013-06-12Bibliographically approved

Open Access in DiVA

No full text

By organisation
Department of Biochemistry
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 522 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf