Synthesis of [3H]phlorizin and its binding behavior to renal brush-border membranes

Tetra- and tribromophlorizin have been synthesized under mild brominating conditions. With catalytic debromination in the presence of hydrogen or tritium gas, bromine atoms in the derivatives were completely substituted by hydrogen or tritium. The product was identical to the native phlorizin and was chemically pure. Tritiated phlorizin with extremely high specific radioactivity (45 Ci/mmol) was obtained when hydrogen gas was replaced by tritium gas. While the brominated compounds showed little inhibition of sodium D-glucose co-transport by isolated renal brush-border membranes. [3H]phlorizin had the same binding affinity to the brush-border membranes as native phlorizin and a Ki value of 1.2 microM for the sodium-dependent D-glucose transport. Binding studies performed using a flow-dialysis method resulted in 150 pmol of phlorizin-binding sites per milligram of membrane protein. This radioactive phlorizin can be a useful tool for determining D-glucose-(phlorizin) binding sites at a low phlorizin concentration in membranes, in nonvesicle forms such as collapsed membrane vesicles, and in purified protein fractions.