Fatty acids, bile acids and non-bile acid cholephils such as bilirubin and sulfobromophthalein (BSP), represent 3 distinct classes of organic anions efficiently extracted by the liver despite tight binding to albumin in the circulation. Sequestration of organic anions is, in fact, a major hepatic function which is vital for survival, but which may be impaired in hepatic injury. This proposal seeks to clarify several aspects of the hepatocellular uptake of organic anions. Specifically, we will investigate (1) whether the transport of ligands bound to albumin is of any particular significance by examining for several carrier proteins (e.g., albumin, Beta-lactoglobulin) the influence of protein concentration, protein: ligand molar ratios and free ligand concentration on specific ligand binding to rat liver sinusoidal plasma membranes, and on ligand uptake in 3 well characterized test systems: the isolated, perfused rat liver, isolated liver cells in suspension or culture, and isolated rat liver plasma membrane vesicles; and (2) whether proteins with high affinities for BSP/bilirubin, fatty acids and bile acids, respectively, recently identified in rat liver sinusoidal plasma membranes, play a significant role in the hepatocellular uptake of their corresponding ligands. Preparative techniques will be improved so as to obtain each of these purified proteins in greater yield, permitting detailed physiochemical characterization. Purified proteins will be employed as antigens for the production of monospecific polyclonal and of monoclonal antibodies. The former will be used in immunofluorescence studies to define the distribution of these membrane proteins on the various cell types within the liver and in non-hepatic tissues. Monoclonal antibodies to the ligand binding site regions of each of the 3 proteins will be identified and their effects on ligand uptake in the 3 test systems described above will be examined. RIA or ELISA assays will be developed and used to seek quantitative correlations between antigen expression and rates of ligand uptake in basal and perturbed states (e.g., phenobarbital, estrogens). If these antigens are identified in other sites (e.g., bile acid binding protein in terminal ileum, fatty acid binding protein in adipocytes) the effect of the appropriate antibody on ligand transport in these sites will also be examined. Finally, if these and other studies suggest a critical role for these proteins in ligand transport, confirmation will be sought by attempting to reconstitute the transport mechanisms in synthetic liposomes.
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