The transport of heme by hemopexin to tissues like liver is a specific, membrane receptor-mediated process. As a result, biologically useful iron is conserved, the accumulation of toxic heme is prevented, and the growth of invading microorganisms inhibited. The ultimate aim of our research is to delineate the biochemical mechanisms of heme transport by hemopexin: from initial binding of heme in the circulation, to the specific interaction with the liver plasma membrane receptor, to transfer of heme from hemopexin to membrane components, and finally to the intracellular transport of heme to heme oxygenase. To attain this goal, a basic approach is being taken in which the components of the hemopexin system are being characterized in detail and their inter-relationships examined to determined the mechanisms by which this system carries out its biological role. This proposal focuses on the hemopexin molecule. the hemopexin receptor and the expression of these proteins in cultured cells. The internalization of the heme and the role of the heme-binding membrane protein (MHBP) discovered in this laboratory are the subject of another project and beyond the scope of this proposal.
The specific aims here are: 1. to define the structure-function relationships of hemopexin with special emphasis on the two domains of hemopexin and on the mechanism of the interaction of hemopexin with irs receptor; 2. to use the polyclonal antibodies to the mouse and human receptor now being prepared to determine the molecular properties of the hemopexin receptor and to clone the receptor cDNA from expression vector libraries; 3. to elucidate the mechanisms which regulate the expression of hemopexin and of its receptor in cultured cells with special attention to the roles of heme and iron in this regulation. Most of the tools needed to achieve these aims are in hand including appropriate lambda gt 11 expression vector libraries, cloned hemopexin cDNA, mono and poly-clonal antibodies to hemopexin and appropriate cell lines. The other needed tools, e.g. antibodies to the receptor, are currently being prepared using the experience gained in earlier stages of this research.
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