Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution

被引:32
作者
Boulter, JM
Wang, DN
机构
[1] NYU, Sch Med, Skirball Inst Biomol Med, New York, NY 10016 USA
[2] NYU, Sch Med, Dept Cell Biol, New York, NY 10016 USA
关键词
D O I
10.1006/prep.2001.1440
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 Angstrom and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degreesC or over 5 weeks at 4 degreesC, This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein. (C) 2001 Academic Press.
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收藏
页码:337 / 348
页数:12
相关论文
共 72 条
  • [1] THE ROLE OF N-GLYCOSYLATION IN THE TARGETING AND STABILITY OF GLUT1 GLUCOSE-TRANSPORTER
    ASANO, T
    TAKATA, K
    KATAGIRI, H
    ISHIHARA, H
    INUKAI, K
    ANAI, M
    HIRANO, H
    YAZAKI, Y
    OKA, Y
    [J]. FEBS LETTERS, 1993, 324 (03) : 258 - 261
  • [2] Baldwin S.A., 1992, MOL ASPECTS TRANSPOR, P169
  • [3] MONOSACCHARIDE TRANSPORTER OF THE HUMAN-ERYTHROCYTE - CHARACTERIZATION OF AN IMPROVED PREPARATION
    BALDWIN, SA
    BALDWIN, JM
    LIENHARD, GE
    [J]. BIOCHEMISTRY, 1982, 21 (16) : 3836 - 3842
  • [4] BALDWIN SA, 1989, METHOD ENZYMOL, V174, P39
  • [5] ERYTHROCYTE METABOLISM - INTERACTION WITH OXYGEN TRANSPORT
    BREWER, GJ
    EATON, JW
    [J]. SCIENCE, 1971, 171 (3977) : 1205 - +
  • [6] MAMMALIAN FACILITATIVE GLUCOSE TRANSPORTERS - EVIDENCE FOR SIMILAR SUBSTRATE RECOGNITION SITES IN FUNCTIONALLY MONOMERIC PROTEINS
    BURANT, CF
    BELL, GI
    [J]. BIOCHEMISTRY, 1992, 31 (42) : 10414 - 10420
  • [7] PROTEOLYTIC AND CHEMICAL DISSECTION OF THE HUMAN-ERYTHROCYTE GLUCOSE TRANSPORTER
    CAIRNS, MT
    ELLIOT, DA
    SCUDDER, PR
    BALDWIN, SA
    [J]. BIOCHEMICAL JOURNAL, 1984, 221 (01) : 179 - 188
  • [8] GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst
    Carayannopoulos, MO
    Chi, MMY
    Cui, Y
    Pingsterhaus, JM
    McKnight, RA
    Mueckler, M
    Devaskar, SU
    Moley, KH
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (13) : 7313 - 7318
  • [9] CARRUTHERS A, 1986, J BIOL CHEM, V261, P1028
  • [10] DETERGENT INTERACTION WITH BAND-3, A MODEL POLYTOPIC MEMBRANE-PROTEIN
    CASEY, JR
    REITHMEIER, RAF
    [J]. BIOCHEMISTRY, 1993, 32 (04) : 1172 - 1179