Proteolytic and chemical dissection of the human erythrocyte glucose transporter.
Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.
D000345:Affinity Labels; D001786:Blood Glucose / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood*; D003571:Cytochalasin B; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006801:Humans; D009004:Monosaccharide Transport Proteins; D013861:Thiocyanates; D014357:Trypsin; D014466:Ultraviolet Rays; D001616:beta-Galactosidase
D000345:Affinity Labels; D001786:Blood Glucose; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D013861:Thiocyanates; D003571:Cytochalasin B; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D014357:Trypsin; C008658:2-nitro-5-thiocyanobenzoic acid