Plasma membrane- and Golgi vesicle-enriched membrane fractions were prepared from day-10 lactating rat mammary glands. Each fraction was found to contain a single set of D-glucose-inhibitable cytochalasin B-binding sites: plasma membranes and Golgi vesicles bound 20 +/- 2 and 53 +/- 4 pmol of cytochalasin/mg of membrane protein (means +/- S.E.M.), with dissociation constants of 259 +/- 47 and 520 +/- 47 nM respectively. Anti-peptide antibodies against the C-terminal region (residues 477-492) of the rat brain/human erythrocyte glucose transporter labelled a sharp band of apparent Mr 50,000 on Western blots of both fractions. Treatment with endoglycosidase F before blotting decreased the apparent Mr of this band to 38,000, indicating that it corresponded to a glycoprotein. Confirmation that this immunologically cross-reactive band was a glucose transporter was provided by the demonstration that it could be photoaffinity-labelled, in a D-glucose-sensitive fashion, with cytochalasin B. Quantitative Western blotting studies yielded values of 28 +/- 5 and 23 +/- 3 pmol of immunologically cross-reactive glucose transporters/mg of membrane protein in the plasma membrane and Golgi vesicle fractions respectively. From comparison with the concentration of cytochalasin B-binding sites, it is concluded that a protein homologous to the rat brain glucose transporter constitutes the major glucose transport species in the plasma membranes of mammary gland epithelial cells. Glucose transporters are also found in the Golgi membranes of these cells, at least half of them being similar, if not identical, to the transporters of the plasma membrane. However, their function in this location remains unclear.
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November 1990
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Research Article|
November 15 1990
Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland
R J Madon;
R J Madon
*Department of Biological Science and Technology, Hannah Research Institute, Ayr KA6 5HL, Scotland, U.K.
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S Martin;
S Martin
*Department of Biological Science and Technology, Hannah Research Institute, Ayr KA6 5HL, Scotland, U.K.
†Departments of Biochemistry, and of Protein and Molecular Biology, Royal Free Hospital School of Medicine (University of London), Rowland Hill Street, London NW3 2PF, U.K.
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A Davies;
A Davies
†Departments of Biochemistry, and of Protein and Molecular Biology, Royal Free Hospital School of Medicine (University of London), Rowland Hill Street, London NW3 2PF, U.K.
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H A C Fawcett;
H A C Fawcett
*Department of Biological Science and Technology, Hannah Research Institute, Ayr KA6 5HL, Scotland, U.K.
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D J Flint;
D J Flint
*Department of Biological Science and Technology, Hannah Research Institute, Ayr KA6 5HL, Scotland, U.K.
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S A Baldwin
S A Baldwin
†Departments of Biochemistry, and of Protein and Molecular Biology, Royal Free Hospital School of Medicine (University of London), Rowland Hill Street, London NW3 2PF, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1990 London: The Biochemical Society
1990
Biochem J (1990) 272 (1): 99–105.
Citation
R J Madon, S Martin, A Davies, H A C Fawcett, D J Flint, S A Baldwin; Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland. Biochem J 15 November 1990; 272 (1): 99–105. doi: https://doi.org/10.1042/bj2720099
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