The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes

Abstract

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.

Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe–lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies

“…As clearly shown in previous work, the receptor discriminates among the three possible conformers of TF ; diferric, the two monoferric species and the apo-protein [10]. Since single lobes of TF either do not bind at all or have limited binding [16,17,26], it is necessary and experimentally more straight forward to use fulllength hTF in binding studies.…”

“…† Data from [10], rabbit serum transferrin binding to rabbit reticulocytes at 4 mC. ‡ Data from [29], used the variant hTF from the patient in England shown to have iron in the N-lobe.…”

“…It is well established that TF receptors discriminate between iron-saturated and iron-free TF as well as the two monoferric species [10]. With a demonstrated difference in the iron-free and iron-saturated Asp mutant proteins in the antibody studies, an experiment was undertaken to determine whether the hTF receptors on HeLa S $ cells would recognize these proteins as ' open ' or ' closed ' conformers.…”

“…iron-free TF [10]. This difference is reflected in the affinity for each conformer of hTF binding to the specific TFR.…”

Immunological study of complex formation between soluble transferrin receptor and transferrin

“…Iron circulates in blood in a complex with a carrier protein, transferrin (Tf) [3,4]. Each TfR subunit binds one transferrin molecule (two transferrin molecules per one molecule of transferrin receptor) [5]. The complex of TfR and ironloaded Tf is transported into cells by means of internalization, where iron is released by pH-dependent mechanism (for details, see [6]).…”