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A biochemical and biophysical investigation into the structure and function of the human erythrocyte glucose transport protein

Davies, Angela Frances; (1991) A biochemical and biophysical investigation into the structure and function of the human erythrocyte glucose transport protein. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

At the onset of this study, much evidence existed for the occurrence of conformational changes in the human erythrocyte glucose transporter, but the regions of the protein affected by these changes were largely unknown. By investigation of the effects of the physiological substrate D-glucose and four inhibitors of glucose transport on the rate of cleavage of the transport protein by trypsin, two hydrophilic cytoplasmically-exposed regions of the polypeptide were implicated in a transport-related conformational change. Further evidence for the occurrence of a confrontational change in the transporter in the presence of one of the transport-inhibitors was also obtained as a result of an investigation by X-ray solution scattering techniques. In order to perform these X-ray solution scattering experiments, it was first necessary to solubilize the transporter in its native form in monodisperse solution. Maintenance of the solubilized transporter in a stable, native form will also be a pre-requisite for future X-ray crystallographic work. The transporter was known to be very unstable when solubilized in octyl glucoside at low temperatures (8°) (Baldwin et al., 1982). Therefore, a range of detergents were investigated for their suitability for solubilization of the glucose transporter. The non-ionic detergent octyl thioglucoside was found to be more efficient than octyl glucoside for solubilization and was also capable of selective solubilization of the glucose transporter as opposed to the nucleoside transporter. Glucose transporter solubilized in either detergent was found to be highly stable if kept at 0°C, more than 75% of the cytochalasin Br-binding activity remaining after 50 hours. However, increasing the temperature of the incubation to 8°C dramatically reduced the stability of the octyl glucoside-solubilized material, only 3% of the cytochalasin B-binding activity remaining after 18 hours. As a result of this work, conditions were found which allowed the study by X-ray solution scattering to be carried out. Identification of the regions of the amino-acid sequence involved in the cytoplasmic and extracellular sugar-binding sites of the glucose transporter would be an important advance in our understanding of the transport mechanism. In this study the glucose transporter was covalently radiolabelled at either the cytoplasmic- or extracellular surface with [3H]cytochalasin B or [3H]2-N-[4-(1-azi -2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-propyl amine ([3H] ATB-BMPA) respectively. Covalent labelling by each of these transport inhibitors is itself inhabitable by Ehglucose, and therefore may involve covalent interactions in the vicinity of the sugar binding-sites at the cytoplasmic and extracellular surfaces respectively. However, one must not dismiss the possibility that the D-glucose- and inhibitor-binding sites are linked allosterically. The radiolabelled protein was then cleaved by a number of enzymatic and chemical means in order to produce a range of fragments, which were identified by the use of antibodies raised against synthetic peptides corresponding to regions of the glucose transporter sequence (Davies et al. ,1987, 1990). This resulted in the identification of two regions of the protein which are likely to be involved in the two sugar-binding sites.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A biochemical and biophysical investigation into the structure and function of the human erythrocyte glucose transport protein
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10116693
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