4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

Abstract

A new phlorizin derivative ( 2′-O-(β- d- glucopyranosyl)-4- azidophloretin , 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na + co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′ i, the constant for fully-competitive inhibition of (Na +, Δψ)-dependent d-glucose uptake, (2) K′ d, the dissociation constant of 4-azido[ 3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″ i, the constant for fully-competitive inhibition of the specific ((Na +, Δψ)-dependent, d-glucose protectable) high-affinity [ 3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants ( K′ i, K′ d and K″ i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′ i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.