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Well-known sugar trasporters: The GLUT1 glucose transporter of human red blood cells and the glucose transporter and lactose permease of Escherichia coli
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2002 (English)In: Recent Research Developments in Biochemistry, Volym 3, Research Signpost, 2002, Vol. 3, 527-546 p.Chapter in book (Refereed)
Place, publisher, year, edition, pages
Research Signpost, 2002. Vol. 3, 527-546 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90676ISBN: 81-7736-155-4 (print)ISBN: 9788177361551 (print)OAI: oai:DiVA.org:uu-90676DiVA: diva2:163120
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. Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
Open this publication in new window or tab >>Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.

The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.

In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 30
Keyword
Biochemistry, Cholesterol, Cytochalasin B, Glucose, GLUT1, Hummel and Dreyer analysis, Immobilization, PEG(5000)-DSPE, Biomembrane, Proteoliposome, Quantitative frontal affinity chromatography, Red blood cell streptavidin-biotin immobilization, Sulfhydryl affinity chromatography, The modified micro-Bradford CaPE assay, MALDI-ToF-MS, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-5727 (URN)91-554-6196-4 (ISBN)
Public defence
2005-04-29, Room B7:113a, BMC, Uppsala, 13:15
Opponent
Supervisors
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2013-06-12Bibliographically approved

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