Binding of Transferrin and Uptake of Iron by Rat Erythroid Cells in Vitro

1. The binding of transferrin and the uptake of iron by rat bone-marrow-cell suspensions was investigated by the use of transferrin doubly labelled with 125I and 59Fe. 2. The pattern of transferrin binding was found to depend on the transferrin concentration in the incubation medium. At relatively low concentrations, binding of transferrin at 0–4 °C was lower than the binding at 37°C. At higher concentrations no difference could be observed between binding at 0–4°C and at 37°C. This phenomenon was explained in terms of a rapid non-specific adsorption of transferrin at 0–4°C, which takes place especially at higher transferrin concentrations, and a specific binding of transferrin at 37°C observed presumably at low concentrations. 3. The maximum number of specific transferrin-binding sites was found to be approximately 190 000 sites per rat reticulocyte and 330 000 sites per nucleated rat bone-marrow cell. The latter number corresponds to 500 000–700 000 sites per nucleated erythroid cell. 4. It was concluded that maturation of the erythroid cell is accompanied with a progressive loss of transferrin binding sites on the cell membrane. 5. When bone-marrow cells obtained after incubation with doubly-labelled transferrin were lysed with distilled water or with the detergent Nonidet P-40, differences in the subcellular distribution of the radioactivities could be observed. 6. It was concluded that the membrane fraction contains appreciable amounts of 59Fe not bound to 125I-labelled transferrin, which indicates that dissociation of the iron—transferrin complex is one of the earlier steps in the mechanism of iron uptake by erythroid cells.