Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes

Abstract

The sidedness of phloretin binding to the glucose carrier has been determined by comparing the type of inhibition produced in zero trans entry and zero trans exit experiments. Initial rates of zero trans entry were measured by the method of R.D. Taverna and R.G. Langdon (Biochim. Biophys. Acta 298:412-421, 1973), which involves pink ghosts loaded with glucose oxidase; this obviates the problem of rapid substrate accumulation inside the cells. With phloretin equilibrated across the membrane, the inhibition of entry was competitive, and the inhibition of exit noncompetitive. The experimental procedures were validated by showing that the inhibition by cytochalasin B, known to bind inside but not outside, was noncompetitive in entry and competitive in exit, as predicted. It was also demonstrated that even after pre-incubation of the cells with a relatively high concentration of phloretin, the phloretin adsorbed in the membrane did not significantly alter the rate of carrier reorientation. The results show that the outward-facing form of the glucose carrier, but not the inward-facing form, bears a phloretin binding site; thus phloretin, as well as cytochalasin B, is bound asymmetrically, phloretin outside and cytochalasin B inside.

Resolution of the direct interaction with and inhibition of the human GLUT1 hexose transporter by resveratrol from its effect on glucose accumulation

“…A secondary plot of the apparent V max values for glucose as a function of resveratrol concentration shows that the inhibition was linear and permits to extrapolate a K i value of 45 M for resveratrol (not shown). As controls we used two known inhibitors that interact with the transporter at either the internal face (cytochalasin B) or the external face (phloretin) (17,27), where only the latter was able to modify the K d value for D-glucose at the external binding site (40,43). Thus our results indicate that resveratrol does not affect the affinity of D-glucose for the external binding site, suggesting that the phytoalexin does not interact with the external face of the transporter.…”

“…Alternatively, on the basis of the obvious structural and chemical differences between these compounds, we favor the option that resveratrol may actually bind to an independent endofacial site whose occupancy renders the carrier refractory to bind cytochalasin B. We propose that resveratrol may elicit the displacement of bound cytochalasin B in a manner similar to the effect of phloretin or genistein, molecules that are known to interact with the external face of the carrier and behave as negative allosteric ligands of cytochalasin B binding (6,27,28,43). In fact, we provided data suggesting that gossypol interacts with an endofacial site on the GLUT1 protein that is distinguishable from the cytochalasin B binding site (44).…”

“…For instance, cytochalasin B binds to the cytosolic face of the transporter and competitively inhibits the exit of glucose from cells (6,17). On the other hand, phloretin binds to the extracellular face GLUT1 and competitively blocks the uptake of glucose (6,27). Despite the wide variety of known GLUT1 inhibitors, most of them show low biological stability or they cannot reach the serum concentration required to exert a significant effect in vivo, which makes necessary the identification of novel blockers that can reach inhibitory concentrations at the level of plasma membranes.…”

Functional characterization of a putative disaccharide membrane transporter in crustacean intestine

“…Phloretin does not have a glucose moiety like phloridzin to bind to the glucose active site on the transporter. Instead, phloretin orients itself on the active site of GLUT2 to prevent glucose or fructose release from the cell (Krupka, 1985). It is being suggested that phloretin acts the same way with the presumptive SCRT-like transporter.…”

A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na+-dependent phosphate absorption

“…Phloridzin is a potent inhibitor of the renal and intestinal BBM Na ϩ -glucose cotransporters (9,36). Phloretin, the aglucone of phloridzin, inhibits a variety of membrane transporters including Band 3 (AE-1) (9, 10) and the facilitated diffusion glucose carrier (GLUT-4) (9,14). Unlike phloridzin, phosphorylated phloretin derivatives are not natural plant products.…”