Phlorizin binding to isolated enterocytes: Membrane potential and sodium dependence

Phlorizin binding is studied in isolated intestinal epithelial cells of the chick. Cells are ATP depleted to allow extensive manipulation of ionic gradients and membrane potential (Δψ). Phlorizin binding is assayed at steady state. Carrier specific phlorizin binding is defined as d-glucose (90 mM) inhibitable binding. Specific binding displays simple Michaelian kinetics as a function of phlorizin. indicating the presence of a single homogeneous binding site. Sodium concentrations and Δψ modify the apparent binding affinity but not the maximum number of binding sites. In contrast, the activation curve as a function of sodium concentrations is sigmoid and the apparent maximum number of binding sites at saturating sodium is phlorizin dependent. The rate of phlorizin association is both Δψ and sodium-concentration dependent. Dissociation is sodium-concentration dependent but not Δψ dependent. Theoretical analysis indicates binding order of substrates is random. In addition, data suggests that the phlorizin/sodium stoichiometry is 2:1. The Δψ dependence can be explained by two models: either translocation is the Δψ-dependent step and the free carrier is anionic, or sodium binding is the Δψ-dependent step.