CELL-SURFACE BIOTINYLATION OF GLUT4 USING BIS-MANNOSE PHOTOLABELS

New cell-impermeant bis-mannose photolabels have been developed with b iotinyl groups attached to l)-benzoyl-1,3-bis(D-mannos-4-yloxy)-2-prop ylamine (ATB-BMPA) by either a polyethoxy sgacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal in hibition constants, K, values, for inhibition of glucose transport act ivity in insulin-stimulated rat adipocytes were determined to be 359 /- 10 and 273 +/- 28 mu M for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respec tively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced c ross-linking of the biotinylated photolabels to rat adipocytes, the bi otinylated 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 mu g of adipose cell me mbranes, precipitated onto magnetic streptavidin beads, could be sensi tively and quantitatively detected using an electrochemiluminescent as say method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent sig nal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 c ould be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotti ng. Data obtained using the non-radioactive methods compared favourabl y with those using tritiated versions of the biotinylated probes. Insu lin treatment of adipocytes increased the levels of signals from surfa ce biotinylated GLUT4 by similar to 10-fold or similar to 20-fold, res pectively, when the electrochemiluminescent or the Western blot detect ion methods were used and these signals were blocked by cytochalasin B .