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Transmembrane exchange of fluorosugars: Characterization of red cell GLUT1 kinetics using 19F NMR

Transmembrane exchange of fluorosugars: Characterization of red cell GLUT1 kinetics using 19F NMR
Transmembrane exchange of fluorosugars: Characterization of red cell GLUT1 kinetics using 19F NMR

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

fluorinated sugar, glucose transport, NMR spectroscopy, erythrocytes, Michaelis-Menten kinetics, enzyme-substrate interactions
0006-3495
Shishmarev, Dmitry
11bac3f9-42c3-42ba-b3d1-e5fc06365580
Fontenelle, Clément Q.
c746c5ea-96bc-46c2-849d-47215ab58c03
Kuprov, Ilya
bb07f28a-5038-4524-8146-e3fc8344c065
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Kuchel, Philip W.
62f434b1-d370-4552-85f4-05b76c625945
Shishmarev, Dmitry
11bac3f9-42c3-42ba-b3d1-e5fc06365580
Fontenelle, Clément Q.
c746c5ea-96bc-46c2-849d-47215ab58c03
Kuprov, Ilya
bb07f28a-5038-4524-8146-e3fc8344c065
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Kuchel, Philip W.
62f434b1-d370-4552-85f4-05b76c625945

Shishmarev, Dmitry, Fontenelle, Clément Q., Kuprov, Ilya, Linclau, Bruno and Kuchel, Philip W. (2018) Transmembrane exchange of fluorosugars: Characterization of red cell GLUT1 kinetics using 19F NMR. Biophysical Journal. (doi:10.1016/j.bpj.2018.09.030).

Record type: Article

Abstract

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

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Accepted/In Press date: 20 September 2018
e-pub ahead of print date: 5 October 2018
Keywords: fluorinated sugar, glucose transport, NMR spectroscopy, erythrocytes, Michaelis-Menten kinetics, enzyme-substrate interactions

Identifiers

Local EPrints ID: 425922
URI: http://eprints.soton.ac.uk/id/eprint/425922
ISSN: 0006-3495
PURE UUID: 43a67703-095b-419e-9eb6-e511b828ba8e
ORCID for Clément Q. Fontenelle: ORCID iD orcid.org/0000-0002-1630-3407
ORCID for Ilya Kuprov: ORCID iD orcid.org/0000-0003-0430-2682
ORCID for Bruno Linclau: ORCID iD orcid.org/0000-0001-8762-0170

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Date deposited: 06 Nov 2018 17:30
Last modified: 26 Nov 2021 06:59

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Contributors

Author: Dmitry Shishmarev
Author: Clément Q. Fontenelle ORCID iD
Author: Ilya Kuprov ORCID iD
Author: Bruno Linclau ORCID iD
Author: Philip W. Kuchel

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