The movement of foreign compounds, in particular the carcinogenic polycyclic aromatic hydrocarbons, through an animal consists of a number of complicated events. The actual or potential steps of metabolic transformation are reasonably well known, however, mechanisms of transport of these highly lipophilic molecules, other than passive diffusion, are virtually unknown. Polycyclics also serve to induce new cytochrome P-450 activity, apparently at the transcriptional level, but again many of the molecular details are unknown. The ultimate fate of these compounds and the actual biological residence time will greatly affect the compounds' carcinogenic properties. Also because they act as inducers of specific isoenzymes of cytochrome P-450 the potential is high for the modulation of their own carcinogenicity or that displayed by other environmental pollutants in either a positive or negative direction. The molecular events of transport and induction then are integral to the understanding of chemical carcinogenesis. Preliminary studies have shown that a protein exists in mouse liver cytosol that binds benzo[a]pyrene with high affinity and in a pharmacologically specific fashion. The concentration of this protein and the dissociation constant (8nM) provide evidence that it may be acting as an intracellular transport protein and/or a receptor involved in cytochrome P-450 induction. The goals in this project are to purify this protein utilizing standard fractionation techniques, photoaffinity labels and affinity chromatography. The structural features important for ligand binding will be studied by competition binding assays and a full kinetic and thermodynamic characterization of the binding process will be studied. By carrying out experiments of nuclear translocation and reversible chemical crosslinking of the cytosol we will probe the nature of this protein as a receptor and will observe molecular aspects of its activation and transformation. The relationship of this protein to the well characterized Ah-receptor, a known receptor for cytochrome P-450 induction, will be investigated. These studies will hopefully provide greater understanding of the carcinogenesis process itself.
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