Both hematopoietic and non-hematopoietic cells require iron for growth and for the synthesis of hemoproteins. Cells take up iron from the plasma glycoprotein transferrin (Tf) after binding of diferric transferrin [Tf(Fe)2] to surface receptors. The subject of this proposal is to study factors regulating receptor number,the rate of internalization of the receptor-ligand complex, the dissociation of iron from Tf(Fe)2 within the cell and the recycling of receptors back to the cell surface. Heme, the end-product of a metabolic pathway utilizing iron, appears to regulate the number of Tf receptors at the level of receptor synthesis. The hypothesis that the intracellular free heme pool regulates receptor synthesis will be tested in several ways. Heme utilization will be altered either by instilling heme binding proteins (apocytochrome C and hemopexin) into the cell cytosol or by inducing apocytochrome P450.n Heme synthesis will be enhanced in normal cells and cells from patients with protoporphyria by supplying substrates for porphyrin biosynthesis. Heme catabolism will be altered either by stimulating or inhibiting heme oxygenase activity. To evaluate the possibility that enhanced catabolism may be matched by enhanced synthesis, resulting in a steady level of intracellular heme, we plan new methods for measuring heme oxygenase activity and substrate flux through the heme biosynthetic pathway. The hypothesis that cells regulate iron uptake, independent of receptor number, by modulating the rate of internalization of Tf(Fe)2 receptor complexes will be tested. Conditions to be studied which may alter ligand internalization include changes in receptor occupancy and contact inhibition of fibroblast cell cultures. Changes in internalization rate may result from altered receptor distribution and approaches are designed to examine this possibility. Iron dissociates from Tf(Fe)2 in an acidic, intracellular vesicle termed the intermediate vesicle. This organelle also contains a large pool of Tf receptors capable of recycling to the cell surface. The intermediate vesicle will be isolated by a unique approach which changes its buoyant density. Monoclonal antibodies will be used to examine the nature and function of the vesicle. The mechanism by which the vesicle """"""""pumps iron"""""""" into the cytosol will be studied with emphasis on defining the cytrosolic iron acceptor. Understanding the regulation of cellular iron uptake should provide insight into alteration of iron uptake which occur in disease states.
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