Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360128
Title: Structural basis of substrate binding to human facilitative sugar transporters
Author: Kane, Susan
ISNI:       0000 0001 3594 5117
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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Abstract:
Human GLUT5 has been heterologously expressed in Xenopus oocytes, allowing examination of its substrate selectivity and basic kinetic parameters. The Km for zero-trans entry of D-fructose by GLUT5 is 22.5 mM, but 2-deoxy-D- glucose is not transported by this isoform. Additionally, transport of fructose is not inhibited by the presence of either deoxyglucose or D-glucose. Analysis of the effects of pH on transport indicate that deoxyglucose is not transported across the pH range. Furthermore, the Km for transport of fructose remains constant under varying pH conditions. The inhibitory effects of fused ring fructose analogues on the fructose transport of GLUT5 have also been determined. Fructose transport by GLUT5- expressing oocytes is not inhibited by the fused pyranose ring analogues, 1,5- anhydromannitol and L-sorbose. However, the fused furanose ring analogue, 2,5 anhydromannitol, does inhibit the transport of fructose by GLUT5 in the same system. GLUT2/GLUT3 chimeras had been constructed previously. Expression of these mutants in Xenopus oocytes has allowed determination of their basic kinetic parameters, with a view to examine the structural basis for differential substrate selectivity between isoforms. Km values for the transport of alternative substrates, fructose and galactose, have been determined for these chimeras, and compared with parameters previously measured for deoxyglucose. All parameters were compared with Km values obtained for native GLUT2 and GLUT3 in the same system. Results indicate that helix 7 participates in fructose recognition by GLUT2, and the equivalent helix in GLUT3 is involved in high affinity deoxyglucose and galactose transport by this isoform. A panel of nineteen GLUT3 mutants had been constructed previously, each incorporating substitution of an individual residue of helix 8 with alanine. These have been expressed in the Xenopus oocyte system, allowing analysis of their basic kinetic properties. The Km value for deoxyglucose entry was determined for each mutant and this compared to that of wild-type GLUT3. Only replacement of asparagine-315 with alanine altered this transport parameter with respect to the wild type value, producing a 4-fold elevation of Km. This residue is polar and may be directly involved in the transport of glucose.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.360128  DOI: Not available
Keywords: Glucose
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