There has been much interest recently in the discovery that certain cells involved in immune responses express membrane sites binding transferrin, an important carrier protein for many metallic cations. The first objective of this project is to perform detailed investigations of the binding of transferrin by trophoblast, lymphoblastoid and other transformed cells, and normal and leukemic lymphocytes, and to obtain further confirmation that these interactions demonstrate the saturability, reversibility, specificity and high affinity required for designation of the binding sites as specific receptors. A combined methodological approach, employing both radioisotopic and immunofluorescense techniques will be used. Secondly, further information concerning the physiology of these binding sites will be obtained by studies of the effects in viable cell suspensions of defined inhibitors of DNA, RNA and protein, and enzymatic treatment with glycosidases, proteases and phospholipases. Thirdly, isolated membranes obtained from cells bearing defined trasferrin receptors will be subjected to solubilization protocols to obtain membrane preparations which will be fractionated to yield purified isolated receptors. This will provide certain physicochemical data concerning transisolated receptors. This will provide certain physicochemical data concerning transferrin binding site structure, and will also permit the production of both monoclonal and conventional heteroantisera which can be used to induce blockade of ligand: receptor interactions, and as probes for further investigations of transferrin receptors. The fourth objective is to study the effects upon binding of transferrin of partial of complete saturation with metallic cations, such as iron and zinc. The subsequent cellular uptake of cation will then be investigated, with particular reference in viable suspensions of cells to the effects of metabolic and cytosketetal inhibitors which appear to inhibit temperature-dependent membrane mobility and endocytosis of transferrin-occupied receptor structures. Fifthly, the relationship between binding of transferrin and the process of activation of peripheral blood lymphocytes will be investigated. These investigations will establish basic parameters of transferrin binding and by demonstrating the cellular disposition of ligand and complexed metallic cations may also indicate possible immumobiological roles of this phenomenon.
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