Cell-surface biotinylation of GLUT4 using bis-mannose photolabels.
New cell-impermeant bis-mannose photolabels have been developed with biotinyl groups attached to 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1, 3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) by either a polyethoxy spacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, Ki values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359+/-10 and 273+/-28 microM for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respectively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced cross-linking of the biotinylated photolabels to rat adipocytes, the biotinylated glucose transporter isoform 4 (GLUT4) could be detected by non-radioactive and radioactive methods that utilized the interaction with streptavidin. Biotinylated GLUT4 from 1-2 microg of adipose cell membranes, precipitated onto magnetic streptavidin beads, could be sensitively and quantitatively detected using an electrochemiluminescent assay method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent signal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 could be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotting. Data obtained using the non-radioactive methods compared favourably with those using tritiated versions of the biotinylated probes. Insulin treatment of adipocytes increased the levels of signals from surface biotinylated GLUT4 by approximately 10-fold or approximately 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.
Koumanov
F
F
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.
Yang
J
J
Jones
A E
AE
Hatanaka
Y
Y
Holman
G D
GD
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D004187:Disaccharides / Q000138:chemical synthesis; D051275:Glucose Transporter Type 4; D007202:Indicators and Reagents; D007328:Insulin / Q000494:pharmacology; D008163:Luminescent Measurements; D008297:Male; D008358:Mannose; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats; D017208:Rats, Wistar; D014466:Ultraviolet Rays
C111925:4,4'-O-(2-(2-(2-(2-(2-(6-(biotinylamino)hexanoyl)amino)ethoxy)ethoxy)ethoxy)-4-(1-azi-2,2,2-trifluoroethyl)benzoyl)amino-1,3-propanediyl bis-mannose; C111924:4,4'-O-(2-(2-(2-(2-(2-(biotinylamino)ethoxy)ethoxy)ethoxy)-4-(1-azi-2,2,2-trifluoroethyl)benzoyl)amino-1,3-propanediyl)bis-mannose; D000345:Affinity Labels; D004187:Disaccharides; D051275:Glucose Transporter Type 4; D007202:Indicators and Reagents; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D003571:Cytochalasin B; D001710:Biotin; D008358:Mannose