We propose to study a major aspect of mammalian copper transport, namely the mechanisms by which cells take up copper from the plasma protein, ceruloplasmin. a major focus of the project is to examine the possibility that ceruloplasmin Cu is delivered by a mechanism involving (1) binding to a cell surface receptor that recognizes the protein and/or carbohydrate moiety of the full Cu loaded molecule; (2) transfer to a Cu binding protein in the membrane; and (3) transfer to a cytosolic carrier, possibly after reduction/re-oxidation. For this, a rapid procedure for isolation of ceruloplasmin, involving immunoaffinity chromatography and preparative electrophoresis, will be sought, allowing isolation of fully Cu loaded, low Cu, and other modified forms of the protein. The presence of specific binding, the kinetics of """"""""on"""""""" and """"""""off"""""""" reactions, and the affinities of receptors, in whole cells and outer (plasma) and internal membranes from a variety of tissues, will be measured with various forms of rat ceruloplasmin labeled singly or doubly with 125I and 67Cu (or in the carbohydrate moiety). The possibility of reductive release of copper from the membrane to cytosolic components will be explored with appropriate chelating and reducing agents. The intracellular pathways the Cu and the protein take after uptake will be followed, using the same radioactive labels, various inhibitors of the uptake processes, subcellular fractionations, and chromatographic/filtration procedures that separate soluble copper components. Isolation and initial characterization of the ceruloplasmin receptor (and putative copper receptor) will be attempted, with appropriate detergent and protein purification techniques plus or minus prior crosslinking. We expect the research to contribute substantially to our basic understanding of the metabolism of an important trace element that has functions in many areas.
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