This proposal seeks to characterize the membrane components involved in the hepatocyte anion transport system, and to elucidate their disposition within the membrane system with the ultimate goal of understanding the architecture, dynamics and regulation of transport channels. Studies using hepatocytes derived from resting and regenerating liver, and hepatoma tissue culture (HTC) cells will be concerned with the analysis of anion transport properties and the identification, isolation and characterization of the membrane associated anion transport proteins using photoaffinity labeling in conjunction wwth differential extraction and chromatographic techniques. The purified proteins will be characterized by amino acid and carbohydrate analysis as well as by tryptic fingerprint mapping. The functional role of these components in the transport process will be evaluated using liposome reconstitution procedures. The proteoliposome system will be evaluated by characterizing sulfate transport properties and the effects of anion transport inhibitors. The structural organization and spacial orientation of the transport components in the hepatocyte and proteoliposome systems will be characterized using a variety of photoaffinity probes in conjunction with proteolytic dissection. The effect of proteolysis of the membrane assocated transport proteins on the transport function of the cells and liposomes will also be evaluated. These studies should provide many insights into the mechanism of hepatocyte anion transport and contribute to our general understanding of transmembrane transport channels.
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