Identification and characterization of the rat adipocyte glucose transporter by photoaffinity crosslinking

The photoaffinity crosslinking agent hydroxysuccinimidyl-4-azidobenzoate has been used to attach [³H]cytochalasin B to a rat adipocyte low-density microsomal membrane protein of 45-50 kDa. The characteristics of the [³H]cytochalasin B-labeled protein are consistent with those of the adipocyte glucose transporter. The low-density microsomes from cells incubated without insulin incorporate twice the amount of radioactivity per mg membrane protein than low-density microsomes derived from insulinstimulated cells. This value agrees with the distribution of glucose transporters measured in this intracellular membrane fraction prepared from basal and insulin-treated cells by [³H]cytochalasin B binding. Preincubation of membranes with 500 mM D-glucose reduces the photoaffinity crosslinking by 48% relative to that observed with 500 mM L-glucose. Isoelectric focusing of low-density microsomes containing the photoaffinity crosslinked transporter yields three bands of radioactivity focusing at pH values of 5.5, 4.5, and 4.2 respectively. Following islation from the isoelectric focusing gel and SDS-polyacrylamide gel electrophoresis, all three peaks can be shown to contain a band of 45-50 kDa which crossreacts with an antiserum raised against the purified human erythrocyte glucose transporter. These results suggest that the identification, isolation and purification of the adipocyte glucose transporter is now possible using the techniques described above.