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Equilibrative nucleoside transporter hENT1 is excluded from raft domains in membranes from normal human erythrocytes
Author(s) -
Craik James,
Sadeq Amal M
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb76-c
Subject(s) - nucleoside transporter , nucleoside , glut1 , glucose transporter , chemistry , red blood cell , biochemistry , staining , membrane , raft , lipid raft , transporter , biology , endocrinology , insulin , polymerization , gene , organic chemistry , polymer , genetics
Nucleoside transport is mediated by hENT1 protein in human red cells and is important both for maintenance of intracellular purine pools and for rapid clearance of adenosine from blood plasma. GLUT1 equilibrative glucose transporter polypeptides show association with both raft and non‐raft domains in a number of cell types and transporter activity of GLUT1 may be subject to acute modification according to its immediate lipid milieu. Since hENT1 polypeptides show strong physical similarities to GLUT1, association of hENT1 with raft domains in human red cell membranes was investigated. Detergent resistant membrane fractions (DRM) were isolated by treatment of red cell membranes with non‐ionic detergents and subsequent ultracentrifugation flotation of fractions on sucrose density gradients. Protein composition of fractions was investigated by SDS‐PAGE (coomassie blue/silver staining) and Western blotting. Treatment with 1% (v/v) Triton X‐100 (4°C) or Brij98 (37°C) gave DRM fractions that contained flotillin1 but not Band 3. Treatment of cells with beta‐methyl‐cyclodextrin substantially decreased yield of DRM fractions. GLUT1 was associated with both DRM and non‐DRM fractions. However, hENT1 could not be detected in DRM fractions from normal red cells using either TX‐100 or Brij98 protocols. Exclusion of hENT1 from ‘raft’ domains points to differences in control of expression and activity of glucose and nucleoside transporter polypeptides in human red cells. Supported by a Research Grant (MB 030) from Kuwait University (JDC) and the College of Graduate Studies, Kuwait University (AS)