Chromatographic fractionation of human reticulocytes after uptake of doubly labelled [59Fe, 125I]transferrin

Abstract

Abstract 1. 1. After exposure to [59Fe, 125I]transferrin, human reticulocytes were separated into membrane (stroma) and water-soluble (cytosol) fractions. 2. 2. The cytosol yielded two 59Fe-bearing components on Sephadex G-200 columns; haemoglobin and a higher molecular weight component which was not further analysed. 3. 3. Membrane solubilised in Triton X-100 was separated by sequential chromatography into three 59Fe-bearing components called A, B1 and B2. Sepharose 2B excluded component A which was thus of high particle size. 4. 4. Passage through Sepharose 2B was an obligatory step in the separation of B1 and B2 from A. Prior to this step. B1 and B2 were not retarded by a Sepharose 4B column whereas after Sepharose 2B both were retarded in Sepharose 4B and also in Sepharose 6B, by which they were more clearly separated. Thus, Sepharose 2B appears to play an active part in the detachment and separation of these components. 5. 5. Components B1 had a molecular weight of the order of 106 and was associated with the main membrane protein. 6. 6. Component B2 had a molecular weight of 230000 and was associated with all the membrane 125I. 7. 7. We conclude that component B2 is a complex of iron transferrin and a membrane binding site. The binding site has a molecular weight about 150000, assuming a 1:1 molar ratio.