Interaction between phloretin and the red blood cell membrane

Abstract

Phloretin binding to red blood cell components has been characterized at pH 6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturable processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that hemoglobin- free red cell ghosts can bind only about 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7 s, corresponding to a permeability coefficient of 2 x 10-4 cm/s. The concentration dependence of the binding to ghosts reveals at least two saturable components. Phloretin binds with high affinity (Adiss = 1.5 /u.M) to about 2.5 x 106 sites per cell; it also binds with lower affinity (Adiss = 54 pM) to a second (5.5 x 107 per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. © 1976, Rockefeller University Press., All rights reserved.