The transport of heme by hemopexin to the liver parenchymal cell is a tissue specific, membrane receptor-mediated process. As a result, biologically useful iron is conserved and accumulation of toxic heme is pervented. The ultimate aim of this research is to delineate the biochemical mechanisms of heme transport by hemopexin from initial binding of heme in the circulation, to 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 taken in which the components of this system are characterized in detail and carefully examined to determine their mechanism of action.
The specific aims for the initial stages of the project are: 1. to continue to define the structure-function relationships of hemopexin using a combination of recombinant DNA techniques, immunochemistry and protein chemistry, and to more fully characterize its heme- and receptor-binding domains; 2. to further delineate the mechanisms of the interaction of hemopexin with its receptor and of heme transfer to the membrane heme binding component (HBC); 3. to determine the molecular properties of the hemopexin receptor and to elucidate how the receptor functions by employing monoclonal antibodies raised to the receptor and affinity chromatography using heme-hemopexin linked to agarose beads; 4. to isolate and characterize the heme binding protein (HBC) found in liver plasma membranes with emphasis on its molecular size, subunit structure, affinity and stoichiometry for heme, and on how the HBC relates to the hemopexin receptor itself. In the subsequent stages of the project, in addition to extending the above work as indicated by on-going results, investigation of the synthesis, regulation and turnover of hemopexin, of the membrane receptor for hemopexin and of HBC will be initiated. The recombinant DNA probes developed to determine the structure of hemopexin and the monoclonal antibodies to the receptor will provide some of the tools needed to probe the metabolism of these proteins in primary cultures of liver hepatocytes and in minimal deviation hepatoma lines. Of special interest is whether the expression of these three proteins is coordinately regulated by heme.
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