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A Micro-Bradford Membrane Protein Assay
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
2000 (Engelska)Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 284, nr 1, s. 162-164Artikel i tidskrift (Refereegranskat) Published
Ort, förlag, år, upplaga, sidor
2000. Vol. 284, nr 1, s. 162-164
Nationell ämneskategori
Naturvetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-92798DOI: 10.1006/abio.2000.4676PubMedID: 10933871OAI: oai:DiVA.org:uu-92798DiVA, id: diva2:166095
Tillgänglig från: 2005-04-07 Skapad: 2005-04-07 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
Ingår i avhandling
1. Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
Öppna denna publikation i ny flik eller fönster >>Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
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
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
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-5727 (URN)91-554-6196-4 (ISBN)
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

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