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Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
2005 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis , 2005. , s. 42
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 30
Nyckelord [en]
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
Nyckelord [sv]
Biokemi
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-5727ISBN: 91-554-6196-4 (tryckt)OAI: oai:DiVA.org:uu-5727DiVA, id: diva2:166100
Disputation
2005-04-29, Room B7:113a, BMC, Uppsala, 13:15
Opponent
Handledare
Tillgänglig från: 2005-04-07 Skapad: 2005-04-07 Senast uppdaterad: 2013-06-12Bibliografiskt granskad
Delarbeten
1. A Micro-Bradford Membrane Protein Assay
Öppna denna publikation i ny flik eller fönster >>A Micro-Bradford Membrane Protein Assay
2000 (Engelska)Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 284, nr 1, s. 162-164Artikel i tidskrift (Refereegranskat) Published
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-92798 (URN)10.1006/abio.2000.4676 (DOI)10933871 (PubMedID)
Tillgänglig från: 2005-04-07 Skapad: 2005-04-07 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
2. Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter
Öppna denna publikation i ny flik eller fönster >>Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter
Visa övriga...
2000 (Engelska)Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, nr 23, s. 6875-6882Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.

Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-92799 (URN)10.1046/j.1432-1033.2000.01788.x (DOI)11082199 (PubMedID)
Tillgänglig från: 2005-04-07 Skapad: 2005-04-07 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
3. On the oligomeric state of the red blood cell glucose transporter GLUT1
Öppna denna publikation i ny flik eller fönster >>On the oligomeric state of the red blood cell glucose transporter GLUT1
2003 (Engelska)Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1618, nr 1, s. 8-16Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

We stripped human red blood cell membranes of cytoskeleton proteins at pH 12 without reductant, partially solubilized the obtained vesicles by use of octaethylene glycol n-dodecyl ether and purified the glucose transporter GLUT1 by anion-exchange chromatography followed by sulfhydryl-affinity chromatography, which removed most of the nucleoside transporter (NT) and the lipids. Eighty percent of the sulfhydryl-bound GLUT1 could be eluted with sodium dodecyl sulfate (SDS) indicating that the bound protein was multimeric. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) of the trypsinized major SDS-PAGE zone of the purified material identified GLUT1 but no other membrane protein. Transmembrane helices 1 and 8 were among the detected fragments. The reconstituted purified GLUT1 showed glucose transport activity, although only approximately 0.05 high-affinity cytochalasin B (CB) binding sites were present per GLUT1 monomer. The vesicles used as starting material for the purification showed 0.4 CB sites per GLUT1 monomer, similar to vesicles prepared in the presence of dithioerythritol. The data are consistent with the coexistence of monomeric GLUT1 with high-affinity CB-binding activity and preferentially solubilized multimeric GLUT1 with no CB-binding activity in the red blood cell membrane vesicles prepared without reductant.

Nyckelord
Chromatography; Affinity, Erythrocytes/*metabolism, Glucose/*metabolism, Glucose Transporter Type 1, Humans, Monosaccharide Transport Proteins/*chemistry/isolation & purification/metabolism, Protein Binding, Protein Conformation, Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization, Structure-Activity Relationship
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
urn:nbn:se:uu:diva-92800 (URN)10.1016/j.bbamem.2003.10.001 (DOI)14643928 (PubMedID)
Tillgänglig från: 2005-04-07 Skapad: 2005-04-07 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
4. The effects of cholesterol and polyethyleneglycol phospholipids on the activities of the reconstituted human red blood cell glucose transporter GLUT1
Öppna denna publikation i ny flik eller fönster >>The effects of cholesterol and polyethyleneglycol phospholipids on the activities of the reconstituted human red blood cell glucose transporter GLUT1
Manuskript (Övrigt vetenskapligt)
Identifikatorer
urn:nbn:se:uu:diva-92801 (URN)
Tillgänglig från: 2005-04-07 Skapad: 2005-04-07 Senast uppdaterad: 2010-01-13Bibliografiskt granskad
5. Well-known sugar trasporters: The GLUT1 glucose transporter of human red blood cells and the glucose transporter and lactose permease of Escherichia coli
Öppna denna publikation i ny flik eller fönster >>Well-known sugar trasporters: The GLUT1 glucose transporter of human red blood cells and the glucose transporter and lactose permease of Escherichia coli
2002 (Engelska)Ingår i: Recent Research Developments in Biochemistry, Volym 3, Research Signpost, 2002, Vol. 3, s. 527-546Kapitel i bok, del av antologi (Refereegranskat)
Ort, förlag, år, upplaga, sidor
Research Signpost, 2002
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-90676 (URN)81-7736-155-4 (ISBN)9788177361551 (ISBN)
Tillgänglig från: 2003-09-04 Skapad: 2003-09-04 Senast uppdaterad: 2013-06-12Bibliografiskt granskad

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