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In order to better understand the cellular delivery of iron from serum transferrin (Tf), we compared iron release from receptor-bound and free Tf. While free Tf did not release all iron until below pH 4.6, receptor-bound Tf released significantly more iron at mildly acidic pH, with essentially all iron released between pH 5.6 and 6.0. Since Tf is acidified to a minimum pH of 5.4 in K562 cells, this result accounts for the nearly complete extraction of iron from Tf by these cells. Comparison of fluorescence from Tf conjugated with lissamine rhodamine sulfonyl chloride (LRSC-Tf) free in solution and bound to receptor provides further evidence that the Tf receptor modulates low pH-mediated conformational changes in Tf. As pH was decreased from neutrality, the fluorescence of free LRSC-Tf began to increase below pH 6.2; the fluorescence of LRSC-Tf bound to human receptors did not increase until below pH 5.6. Binding to the Tf receptor, while facilitating iron release from Tf, appears to partially inhibit a conformational change that causes the increase in LRSC-Tf fluorescence at low pH. The fluorescence of human LRSC-Tf bound to murine receptors increases at a higher pH, 6.0, indicating that there are differences in conformational stabilization of Tf by receptors of different species. The results suggest that the Tf receptor, in addition to providing a means by which cells may internalize Tf, functions to increase the release of iron from Tf in the endosome.

Transferrin (Tf) acidification has been shown to be limited to pH 6 in murine Balb/c 3T3 fibroblasts, human A549 epidermoid carcinoma cells, and Chinese hamster ovary cells and is followed by alkalinization during recycling. In contrast, Tf acidification in the human erythroleukemic cell line K562 proceeds to below pH 5.5, and alkalinization of internal Tf during recycling is not observed. To explore the regulation of endosomal pH in K562 cells, we determined whether the existence of an early endosome of pH 6 could be demonstrated in K562 cells. The kinetics of Tf internalization, acidification, and recycling were determined at temperatures which block recycling of Tf in 3T3 cells. As in 3T3, Tf recycling in K562 was inhibited at 24 degrees C and below. At these temperatures, Tf internalization and acidification were delayed relative to 37 degrees C, yet the minimum pH achieved was below 5.5. Temperatures at or below 19 degrees C resulted in a complete block in recycling (at least over 40 min), which was rapidly reversible by incubation at 37 degrees C. Ouabain (a specific inhibitor of the Na+,K(+)-ATPase) had no effect on K562 Tf acidification, indicating that K562 endosomal pH is probably not regulated by the Na+,K(+)-ATPase. The results suggest that differentiation of the early endocytic pathway in erythroid cells involves changes such that the pH of Tf-containing compartments is not limited to 6 by the Na+,K(+)-ATPase.