Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection.

摘要: Glucose transporter proteins (zone 4.5) which had been photoaffinity labeled with [3H]cytochalasin B in human erythrocyte ghosts were subjected to enzymatic dissection order study the transmembrane disposition of protein situ. Proteolytic enzymes as well glycosidases used treat unsealed and resealed explore various membrane domains a topographically defined manner. Limited digestion sealed trypsin no effect on apparent Mr (55,000). Similar treatment ghosts, however, resulted generation major fragment 21.5 kDa, along several minor fragments. Thermolysin also but caused complete loss radiolabel from zone 4.5 region lower-molecular-weight fragments being retained gel. Chymotrypsin single peak, = 18,400, both indicating its action occurs at outer surface. Digestion carboxypeptidase aminopeptidase indicate C-terminal end is located exterior N terminus cytoplasmic Treatment endoglycosidase shift mobility lower 49,000. The results obtained that carbohydrate near cytochalasin B-binding site N-terminal end.