Kinetics of iron passage through subcellular compartments of rabbit reticulocytes.

The kinetics of the separate processes of Fe2(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and 59Fe, 125I-labeled rabbit transferrin. The binding of 59Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2 x 10(5) M-1 sec-1. The rate of internalization of 59Fe-transferrin was directly measured at 520 +/- 100 molecules of Fe2(III)-transferrin internalized/sec/cell with 250 +/- 43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe2(III)-transferrin internalization the flux of 59Fe was followed through three compartments: internalized transferrin, membrane, and cytosol. A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17 +/- 2 sec and 34 +/- 5 sec, respectively. Apparent rate constants of 0.0075 +/- 0.002 sec-1 and 0.0343 +/- 0.0118 sec-1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112 +/- 0.0025 sec-1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe2(III)-transferrin that are distinct from the handling of transferrin.