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Hydrophobic photolabeling in membranes: the human erythrocyte glucose transporter
Author(s) -
Lala AK,
Bhat S.
Publication year - 1990
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1990.tb00133.x
Subject(s) - band 3 , sialoglycoproteins , chemistry , chromatography , membrane , biochemistry , transporter , glucose transporter , octyl glucoside , membrane protein , gel electrophoresis , polyacrylamide gel electrophoresis , reagent , sodium dodecyl sulfate , biology , enzyme , insulin , gene , endocrinology
Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2‐[3H]Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene‐labeled glucose transporter, the membrane preparation was solubilized with Triton X‐100 and passed through a DEAE‐cellulose column. The flow‐through fraction was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow‐through fraction. On the other hand, use of n‐octyl glucoside gave a relatively better preparation. The 2‐[3H]DAF‐labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C‐terminal transmembrane fragment is labeled.